阅读说明：本技术 治疗神经内分泌肿瘤的方法 (Method of treating neuroendocrine tumors ) 是由 戴伦·西格尔 于 2018-08-21 设计创作，主要内容包括：本文公开了治疗有需要的个体的神经内分泌肿瘤(NET)的方法,包括向个体施用治疗有效量的抑制原肌球蛋白受体激酶(Trk)蛋白的药剂,其中NET与经历遗传易位的Trk蛋白或为NTRK基因融合蛋白的Trk蛋白相关。本文还公开了治疗有需要的个体的神经内分泌肿瘤(NET)的方法,包括：从个体获得NET遗传物质的样品；确定NET肿瘤是否包含NTRK易位或基因融合；以及向个体施用治疗有效量的抑制原肌球蛋白受体激酶(Trk)蛋白的药剂。(Disclosed herein are methods of treating a neuroendocrine tumor (NET) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an agent that inhibits a tropomyosin-receptor-kinase (Trk) protein, wherein the NET is associated with a Trk protein that undergoes a genetic translocation or a Trk protein that is an NTRK gene fusion protein. Also disclosed herein are methods of treating a neuroendocrine tumor (NET) in a subject in need thereof, comprising: obtaining a sample of NET genetic material from an individual; determining whether NET tumors comprise an NTRK translocation or gene fusion; and administering to the subject a therapeutically effective amount of an agent that inhibits a tropomyosin-receptor-kinase (Trk) protein.)

1. A method of treating a neuroendocrine tumor (NET) in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of an agent that inhibits a tropomyosin-receptor-kinase (Trk) protein, wherein the NET is associated with (1) a Trk fusion protein or (2) an NTRK gene that undergoes a genetic translocation.

2. The method of claim 1, wherein the Trk fusion protein comprises a TrkA, TrkB or TrkC tyrosine kinase signaling domain.

3. The method of claim 2, wherein the Trk fusion protein is constitutively active.

4. The method of claim 1, wherein the Trk fusion protein comprises:

(a) an N-terminal polypeptide region comprising the sequence of a polypeptide other than a TrkA, TrkB or TrkC polypeptide; and

(b) a C-terminal polypeptide region comprising the sequence of a TrkA, TrkB or TrkC polypeptide, wherein the C-terminal polypeptide region has TrkA, TrkB or TrkC kinase activity.

5. The method of claim 1, wherein the Trk fusion protein comprises a nucleic acid sequence comprising:

(a) a first region corresponding to a sequence from the MPRIP, CD74, RABGAP1L, TPM3, TPR, TFG, PPL, CHTOP, ARHGEF2, NFASC, BCAN, LMNA, TP53, QKI, NACC2, VCL, AGBL4, TRIM24, PAN3, AFAP1, SQSTM1, ETV6, BTBD1, LYN, RBPMS, RFWD2, IRF2BP2, SSBP2, C18ORF8, RNF213, TBC1, DNEKHA 6, PEAR1, MRPL24, MDM4, GRIPAP1, EPS15, DYNC2H1, CEL, EPHB2, EML4, HOMER2, TEL or FAT1 gene sequence; and

(b) a second region corresponding to an NTRK1, NTRK2, or NTRK3 gene sequence.

6. The method of claim 5, wherein the NTRK gene fusion protein is an ETV6-NTRK gene fusion protein.

7. The method of claim 1, wherein the Trk fusion protein comprises:

(a) an N-terminal polypeptide region comprising the sequence of an MPRIP, CD74, RABGAP1L, TPM3, TPR, TFG, PPL, CHTOP, ARHGEF2, NFASC, BCAN, LMNA, TP53, QKI, NACC2, VCL, AGBL4, TRIM24, PAN3, AFAP1, SQSTM1, ETV6, BTBD1, LYN, RBPMS, RFWD2, IRF2BP2, SSBP2, C18ORF8, RNF213, TBC1, DNEKHA 6, PEAR1, MRPL24, MDM4, GRIPAP1, EPS15, DYNC2H1, CEL, EPHB2, EML4, HOMER2, TEL or FAT1 polypeptide; and

(b) a C-terminal polypeptide region comprising the sequence of a TrkA, TrkB or TrkC polypeptide, wherein the C-terminal polypeptide region has TrkA, TrkB or TrkC kinase activity.

8. The method of claim 7, wherein the N-terminal polypeptide region comprises an ETV6 polypeptide sequence.

9. The method of claim 8, wherein said C-terminal polypeptide region comprises a TrkC polypeptide sequence.

10. The method of claim 1, wherein the Trk fusion protein comprises:

(a) an N-terminal polypeptide region comprising the sequence of a polypeptide other than a TrkA, TrkB or TrkC polypeptide; and

(b) a C-terminal polypeptide region comprising the sequence of a TrkA polypeptide, wherein the C-terminal polypeptide comprises TrkA kinase activity; and is

Wherein the fusion protein is TP53-TrkA, LMNA-TrkA, CD74-TrkA, TFG-TrkA, TPM3-TrkA, NFASC-TrkA, BCAN-TrkA, MPRIP-TrkA, TPR-TrkA, RFWD2-TrkA, IRF2BP2-TrkA, SQSTM1-TrkA, SSBP2-TrkA, RABGAP1L-TrkA, C18ORF8-TrkA, RNF213-TrkA, TBC1D22A-TrkA, C200RF112-TrkA, DNER-TrkA, ARHGEF2-TrkA, CHTOP-TrkA, PPL-TrkA, PLEKHA 2-TrkA, PEAR1-TrkA, MRPL24-TrkA, MDM4-TrkA, TRRC 8-TrkA, GRAP 6-TrkA, GRLR 6-TrkA, TrkYN A, EPC 27-TrkA, EPWD 27-TrkA or EPWD-TrkA fusion protein.

11. The method of claim 1, wherein the Trk fusion protein comprises:

(a) an N-terminal polypeptide region comprising the sequence of a polypeptide other than a TrkA, TrkB or TrkC polypeptide; and

(b) a C-terminal polypeptide region comprising the sequence of a TrkB polypeptide, wherein the C-terminal polypeptide comprises TrkB kinase activity; and is

Wherein the fusion protein is NACC2-TrkB, QKI-TrkB, AFAP1-TrkB, PAN3-TrkB, SQSTM1-TrkB, TRIM24-TrkB, VCL-TrkB, AGBL4-TrkB or DAB2IP-TrkB fusion protein.

12. The method of claim 1, wherein the Trk fusion protein comprises:

(a) an N-terminal polypeptide region comprising a polypeptide sequence other than a TrkA, TrkB or TrkC polypeptide sequence; and

(b) a C-terminal polypeptide region comprising the sequence of a TrkC polypeptide, wherein the C-terminal polypeptide comprises TrkC kinase activity; and is

Wherein the fusion protein is an ETV6-TrkC, BTBD1-TrkC, LYN-TrkC, RBPMS-TrkC, EML4-TrkC, HOMER2-TrkC, TFG-TrkC, FAT1-TrkC or TEL-TrkC fusion protein.

13. The method of claim 1, wherein the agent that inhibits a Trk protein is a kinase inhibitor.

14. The method according to claim 1, wherein the agent that inhibits the Trk protein is emtricinib, RXDX-102, erlotinib, larotinib, LOXO-195, cetritinib, cabozantinib, mellitinib, dorivitinib, crizotinib, TSR-011, DS-6051, PLX7486, lestatinib, darussertib, F17752, AZD6918, AZD7451, or AZ-23, or a pharmaceutically acceptable salt thereof.

15. The method of claim 14, wherein the agent is emtricinib or a pharmaceutically acceptable salt thereof.

16. The method of claim 1, wherein the NET is a foregut NET, a midgut NET or a hindgut NET.

17. The method of claim 1, wherein the NETs are gastrointestinal NETs.

18. The method of claim 1, wherein the NET is a small intestine NET (SI-NET).

19. The method of claim 1, wherein the NET is a large intestine NET.

20. The method of claim 1, wherein the NET is a rectal NET.

21. The method of claim 1, wherein the NET is a gastrointestinal NET.

22. The method of claim 1, wherein the NET is pancreatic NET (pnet).

23. The method of claim 1, wherein the NET is a bronchial NET.

24. The method of claim 1, wherein the NET is appendiceal NET, ovarian NET or thyroid NET.

25. The method of claim 1, wherein the original origination source of the NET is unknown.

26. The method of claim 1, wherein the primary NET has been transferred to a secondary tissue.

27. The method of claim 26, wherein the secondary tissue is lymph node, mesentery, liver, bone, lung, or brain.

28. The method of claim 1, wherein the agent is used as a primary or first line therapy.

29. The method of claim 1, wherein the agent is used as a secondary or rescue therapy.

30. The method of claim 1, wherein the subject has a stable or progressive disease following a previous chemotherapeutic treatment regimen.

31. The method of claim 30, wherein the prior chemotherapy treatment regimen comprises treatment with capecitabine, 5-fluorouracil, doxorubicin, etoposide, dacarbazine, streptozotocin, temozolomide, cisplatin, cyclophosphamide, thalidomide, or any combination thereof.

32. The method of claim 1, further comprising administering additional therapy to the individual.

33. The method of claim 32, wherein said additional therapy is a second agent that inhibits the Trk protein.

34. The method of claim 32, wherein the additional therapy comprises a PI3K/Akt/mTOR pathway inhibitor, a TGF-agent pathway inhibitor, a cell cycle inhibitor, a somatostatin analog, an interferon, or an angiogenesis inhibitor.

35. The method of claim 34, wherein the somatostatin analog is octreotide, octreotide acid, pasireotide, or lanreotide.

36. The method according to claim 34, wherein said somatostatin analog is radiolabeled.

37. The method of claim 37, wherein the radiolabeled somatostatin analog is [ DOTA ™ ]⁰,Tyr³]Octreotide acid (lutetium oxo octreotide).

38. The method of claim 34, wherein the interferon is a type I interferon.

39. The method of claim 38, wherein the type I interferon is IFN- α.

40. The method of claim 34, wherein the adjunctive therapy is everolimus, temsirolimus, bevacizumab, sunitinib, or sorafenib.

41. The method of claim 32, wherein the additional therapy comprises surgery, chemotherapy, or radiation therapy.

42. The method of claim 1, wherein the individual has carcinoid syndrome.

43. The method of claim 42, further comprising administering to the individual a treatment for the carcinoid syndrome.

44. The method of claim 43, wherein the treatment for the carcinoid syndrome is an anti-5-hydroxytryptamine agent.

45. The method of claim 44, wherein the anti-5-hydroxytryptamine agent is a somatostatin analog.

46. The method according to claim 45, wherein said somatostatin analog is octreotide, octreotide acid, pasireotide, or lanreotide.

47. A method of treating a neuroendocrine tumor (NET) in a subject in need thereof, comprising: (a) providing a sample of NET genetic material from the individual; (b) determining whether the NET tumor comprises an NTRK translocation or gene fusion; and (c) administering to the individual an agent that inhibits a tropomyosin-receptor-kinase (Trk) protein.

48. The method of claim 47, wherein the sample of NET genetic material is total nucleic acid obtained from a Formalin Fixed Paraffin Embedded (FFPE) tumor biopsy sample.

49. The method of claim 47, further comprising sequencing at least a portion of the NET genetic material to determine whether the tumor sample comprises an NTRK translocation or gene fusion.

50. The method of claim 49, wherein the NET genetic material is sequenced by whole genome DNA sequencing, whole exome sequencing, targeted DNA sequencing, targeted RNA sequencing, or whole transcriptome RNA sequencing.

51. The method of claim 47, further comprising amplifying the NET genetic material using NTRK1, NTRK2, or NTRK3 specific primers prior to determining whether the NET tumor comprises an NTRK translocation or gene fusion.

52. The method of claim 48, further comprising detecting the amount of NTRK expression or Trk protein level in a NET tumor sample, wherein an increase in NTRK expression level or Trk protein level is indicative of an NTRK translocation or gene fusion.

Background

The median survival of patients with metastatic neuroendocrine tumors (NET) is only 33 months with a poorer prognosis than previously expected. It has been reported that the incidence of NET has increased by five fold over the last thirty years, with prevalence rates of 35 in 100,000 points, and that diagnosis and treatment of NET has become an unmet significant medical need.

Disclosure of Invention

Disclosed herein, in some embodiments, are methods of treating a neuroendocrine tumor (NET) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an agent that inhibits a tropomyosin-receptor-kinase (Trk) protein, wherein the NET is associated with a Trk protein that undergoes genetic translocation or a Trk protein that is an NTRK gene fusion protein. In some embodiments, the NTRK gene fusion protein comprises an NTRK1, NTRK2, or NTRK3 tyrosine kinase signaling domain. In some embodiments, the NTRK gene fusion protein is constitutively active. In some embodiments, the NTRK gene fusion protein comprises: (a) an N-terminal polypeptide region comprising a polypeptide sequence other than a TrkA, TrkB or TrkC polypeptide sequence; and (b) a C-terminal polypeptide region comprising a TrkA, TrkB or TrkC polypeptide sequence, wherein the C-terminal polypeptide region has TrkA, TrkB or TrkC kinase activity. In some embodiments, the NTRK gene fusion protein comprises a nucleic acid sequence comprising: (a) a first region corresponding to a sequence from the MPRIP, CD74, RABGAP1L, TPM3, TPR, TFG, PPL, CHTOP, ARHGEF2, NFASC, BCAN, LMNA, TP53, QKI, NACC2, VCL, AGBL4, TRIM24, PAN3, AFAP1, SQSTM1, ETV6, BTBD1, LYN, RBPMS, RFWD2, IRF2BP2, SSBP2, C18ORF8, RNF213, TBC1, DNEKHA 6, PEAR1, MRPL24, MDM4, GRIPAP1, EPS15, DYNC2H1, CEL, EPHB2, EML4, HOMER2, TEL or FAT1 gene sequence; and (b) a second region corresponding to an NTRK1, NTRK2, or NTRK3 gene sequence.

In the above methods disclosed herein, in some embodiments, the NTRK gene fusion protein is an ETV6-NTRK gene fusion protein. In some embodiments, the NTRK gene fusion protein comprises: (a) an N-terminal polypeptide region comprising the sequence of an MPRIP, CD74, RABGAP1L, TPM3, TPR, TFG, PPL, CHTOP, ARHGEF2, NFASC, BCAN, LMNA, TP53, QKI, NACC2, VCL, AGBL4, TRIM24, PAN3, AFAP1, SQSTM1, ETV6, BTBD1, LYN, RBPMS, RFWD2, IRF2BP2, SSBP2, C18ORF8, RNF213, TBC1, DNEKHA 6, PEAR1, MRPL24, MDM4, GRIPAP1, EPS15, DYNC2H1, CEL, EPHB2, EML4, HOMER2, TEL or FAT1 polypeptide sequence; and (b) a C-terminal polypeptide region comprising a TrkA, TrkB or TrkC polypeptide sequence, wherein the C-terminal polypeptide region has TrkA, TrkB or TrkC kinase activity. In some embodiments, the N-terminal polypeptide region comprises an ETV6 polypeptide sequence. In some embodiments, the C-terminal polypeptide region comprises a TrkC polypeptide sequence. In some embodiments, the NTRK gene fusion protein comprises: (a) an N-terminal polypeptide region comprising a polypeptide sequence other than a TrkA, TrkB or TrkC polypeptide sequence; and (b) a C-terminal polypeptide region comprising a TrkA polypeptide sequence, wherein the C-terminal polypeptide comprises TrkA kinase activity; wherein the fusion protein is TP53-TrkA, LMNA-TrkA, CD74-TrkA, TFG-TrkA, TPM3-TrkA, NFASC-TrkA, BCAN-TrkA, MPRIP-TrkA, TPR-TrkA, RFWD2-TrkA, IRF2BP2-TrkA, SQSTM1-TrkA, SSBP2-TrkA, RABGAP1L-TrkA, C18ORF8-TrkA, RNF213-TrkA, TBC1D22A-TrkA, C200RF112-TrkA, DNER-TrkA, ARHG 2-TrkA, CHTOP-TrkA, PPL-TrkA, PLEKHA 2-TrkA, PEAR1-TrkA, MRPL24-TrkA, MDM4-TrkA, TrkRC 8-TrkA, GRLRAP 6-TrkA, TrkIPHA 2-TrkA, EPLTrkYN 27-TrkA, EPLTrkA or EPLTrkYN 27-TrkA. In some embodiments, the NTRK gene fusion protein comprises: (a) an N-terminal polypeptide region comprising a polypeptide sequence other than a TrkA, TrkB or TrkC polypeptide sequence; and (b) a C-terminal polypeptide region comprising a TrkB polypeptide sequence, wherein the C-terminal polypeptide comprises TrkB kinase activity; wherein the fusion protein is NACC2-TrkB, QKI-TrkB, AFAP1-TrkB, PAN3-TrkB, SQSTM1-TrkB, TRIM24-TrkB, VCL-TrkB, AGBL4-TrkB or DAB2IP-TrkB fusion protein. In some embodiments, the NTRK gene fusion protein comprises: (a) an N-terminal polypeptide region comprising a polypeptide sequence other than a TrkA, TrkB or TrkC polypeptide sequence; and (b) a C-terminal polypeptide region comprising a TrkC polypeptide sequence, wherein the C-terminal polypeptide comprises TrkC kinase activity; wherein the fusion protein is ETV6-TrkC, BTBD1-TrkC, LYN-TrkC, RBPMS-TrkC, EML4-TrkC, HOMER2-TrkC, TFG-TrkC, FAT1-TrkC or TEL-TrkC fusion protein.

In the above methods disclosed herein, in some embodiments, the agent that inhibits the Trk protein is a kinase inhibitor. In some embodiments, the agent that inhibits the Trk protein is emtricinib (entretinib), RXDX-102, erlotinib (altiratinib), larotinib (larotretinib), LOXO-195, cetrortinib (sitravatinib), cabozantinib (cabozantinib), meletinib (merestinib), dolivitinib (dovidinib), crizotinib (crizotinib), TSR-011, DS-6051, PLX7486, lestatinib (lestatatinib), darussertib (danutib), F17752, AZD6918, AZD7451, or AZ-23, or a pharmaceutically acceptable salt thereof. In some embodiments, the agent that inhibits the Trk protein is enretinib. In some embodiments, NET is foregut NET, midgut NET or hindgut NET. In some embodiments, the NET is a gastrointestinal (gastroenterological) NET. In some embodiments, the NET is a small intestine NET (SI-NET). In some embodiments, the NET is a large intestine NET. In some embodiments, the NET is a rectal NET. In some embodiments, the NET is a gastrointestinal (gastrointestinal) NET. In some embodiments, NET is pancreatic NET (pnet). In some embodiments, NET is bronchial NET. In some embodiments, the NET is appendiceal NET, ovarian NET or thyroid NET. In some embodiments, the original origination source of the NET is unknown. In some embodiments, the primary NET has been transferred to a secondary tissue. In some embodiments, the secondary tissue is lymph nodes, mesentery, liver, bone, lung, or brain. In some embodiments, the agent is used as a primary or first line therapy. In some embodiments, the agent is used as a secondary or rescue therapy. In some embodiments, the subject has stable or progressive disease following a previous chemotherapy treatment regimen. In some embodiments, the prior chemotherapeutic treatment regimen comprises treatment with capecitabine (capecitabine), 5-fluorouracil (5-fluoroouracil), doxorubicin (doxorubicin), etoposide (etoposide), dacarbazine (dacarbazine), streptozotocin (streptozocin), temozolomide (temozolomide), cisplatin (cilaspsin), cyclophosphamide (cyclophosphamide), thalidomide (thalidomide), or any combination thereof.

In some embodiments, the above-described methods disclosed herein further comprise administering to the individual additional therapy in some embodiments, the additional therapy is a second agent that inhibits the Trk protein in some embodiments, the additional therapy comprises a PI3K/Akt/mTOR pathway inhibitor, a TGF- β pathway inhibitor, a cell cycle inhibitor, a cell growth factor, a tumor suppressorAn inhibitor, a somatostatin analog, an interferon, or an angiogenesis inhibitor. In some embodiments, the somatostatin analog is octreotide (octreotide), octreotate (octreotate), pasireotide (pasireotide), or lanreotide (lanreotide). In some embodiments, the somatostatin analog is radiolabeled. In some embodiments, the radiolabeled somatostatin analog is [ DOTA ]⁰,Tyr³]In some embodiments, the additional therapy is everolimus (everolimus), temsirolimus (temsirolimus), bevacizumab (bevacizumab), sunitinib (sunitinib), or sorafenib (sorafenib). in some embodiments, the additional therapy comprises surgery, chemotherapy, or radiation therapy.

In some embodiments, disclosed herein are methods of treating a neuroendocrine tumor (NET) in a subject in need thereof, comprising: (a) obtaining a sample of NET genetic material from an individual; (b) determining whether NET tumors comprise an NTRK translocation or gene fusion; and (c) administering to the subject an agent that inhibits a tropomyosin-receptor-kinase (Trk) protein. In some embodiments, the sample of NET genetic material is total nucleic acid obtained from a Formalin Fixed Paraffin Embedded (FFPE) tumor biopsy sample. In some embodiments, the methods disclosed herein further comprise sequencing NET genetic material to determine whether the tumor sample comprises an NTRK translocation or gene fusion. In some embodiments, the NET genetic material is sequenced by whole genome DNA sequencing, whole exome sequencing, targeted DNA sequencing, targeted RNA sequencing, or whole transcriptome RNA sequencing. In some embodiments, the methods disclosed herein further comprise amplifying NET genetic material using NTRK1, NTRK2, or NTRK 3-specific primers prior to determining whether NET tumors comprise an NTRK translocation or gene fusion. In some embodiments, the methods disclosed herein further comprise detecting an amount of NTRK expression or a level of Trk protein in a NET tumor sample, wherein an increase in the level of NTRK expression or the level of Trk protein is indicative of an NTRK translocation or gene fusion.

Also disclosed herein, in some embodiments, is a method of treating a Gastrointestinal (GI) neuroendocrine tumor (NET) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an agent that inhibits a tropomyosin-receptor-kinase (Trk) protein, wherein the NET is associated with a Trk protein that undergoes genetic translocation or a Trk protein that is an NTRK gene fusion protein. In some embodiments, the NTRK gene fusion protein comprises an NTRK1, NTRK2, or NTRK3 tyrosine kinase signaling domain. In some embodiments, the NTRK gene fusion protein is constitutively active. In some embodiments, the NTRK gene fusion protein comprises: (a) an N-terminal polypeptide region comprising a polypeptide sequence other than a TrkA, TrkB or TrkC polypeptide sequence; and (b) a C-terminal polypeptide region comprising a TrkA, TrkB or TrkC polypeptide sequence, wherein the C-terminal polypeptide region has TrkA, TrkB or TrkC kinase activity. In some embodiments, the NTRK gene fusion protein comprises: (a) an N-terminal polypeptide region comprising the sequence of an MPRIP, CD74, RABGAP1L, TPM3, TPR, TFG, PPL, CHTOP, ARHGEF2, NFASC, BCAN, LMNA, TP53, QKI, NACC2, VCL, AGBL4, TRIM24, PAN3, AFAP1, SQSTM1, ETV6, BTBD1, LYN, RBPMS, RFWD2, IRF2BP2, SSBP2, C18ORF8, RNF213, TBC1, DNEKHA 6, PEAR1, MRPL24, MDM4, GRIPAP1, EPS15, DYNC2H1, CEL, EPHB2, EML4, HOMER2, TEL or FAT1 polypeptide sequence; and (b) a C-terminal polypeptide region comprising a TrkA, TrkB or TrkC polypeptide sequence, wherein the C-terminal polypeptide region has TrkA, TrkB or TrkC kinase activity. In some embodiments, the N-terminal polypeptide region comprises an ETV6 polypeptide sequence. In some embodiments, the C-terminal polypeptide region comprises a TrkC polypeptide sequence.

In the above methods disclosed herein for treating GI-NET, in some embodiments, the NTRK gene fusion protein comprises: (a) an N-terminal polypeptide region comprising a polypeptide sequence other than a TrkA, TrkB or TrkC polypeptide sequence; and (b) a C-terminal polypeptide region comprising a TrkA polypeptide sequence, wherein the C-terminal polypeptide comprises TrkA kinase activity; wherein the fusion protein is TP53-TrkA, LMNA-TrkA, CD74-TrkA, TFG-TrkA, TPM3-TrkA, NFASC-TrkA, BCAN-TrkA, MPRIP-TrkA, TPR-TrkA, RFWD2-TrkA, IRF2BP2-TrkA, SQSTM1-TrkA, SSBP2-TrkA, RABGAP1L-TrkA, C18ORF8-TrkA, RNF213-TrkA, TBC1D22A-TrkA, C200RF112-TrkA, DNER-TrkA, ARHG 2-TrkA, CHTOP-TrkA, PPL-TrkA, PLEKHA 2-TrkA, PEAR1-TrkA, MRPL24-TrkA, MDM4-TrkA, TrkRC 8-TrkA, GRLRAP 6-TrkA, TrkIPHA 2-TrkA, EPLTrkYN 27-TrkA, EPLTrkA or EPLTrkYN 27-TrkA. In some embodiments, the NTRK gene fusion protein comprises: (a) an N-terminal polypeptide region comprising a polypeptide sequence other than a TrkA, TrkB or TrkC polypeptide sequence; and (b) a C-terminal polypeptide region comprising a TrkB polypeptide sequence, wherein the C-terminal polypeptide comprises TrkB kinase activity; wherein the fusion protein is NACC2-TrkB, QKI-TrkB, AFAP1-TrkB, PAN3-TrkB, SQSTM1-TrkB, TRIM24-TrkB, VCL-TrkB, AGBL4-TrkB or DAB2IP-TrkB fusion protein. In some embodiments, the NTRK gene fusion protein comprises: (a) an N-terminal polypeptide region comprising a polypeptide sequence other than a TrkA, TrkB or TrkC polypeptide sequence; and (b) a C-terminal polypeptide region comprising a TrkC polypeptide sequence, wherein the C-terminal polypeptide comprises TrkC kinase activity; wherein the fusion protein is ETV6-TrkC, BTBD1-TrkC, LYN-TrkC, RBPMS-TrkC, EML4-TrkC, HOMER2-TrkC, TFG-TrkC, FAT1-TrkC or TEL-TrkC fusion protein. In some embodiments, the agent that inhibits the Trk protein is a kinase inhibitor. In some embodiments, the agent that inhibits the Trk protein is emtricinib, RXDX-102, erlotinib, LOXO-195, cetrortinib, cabozantinib, mellitinib, doxertinib, crizotinib, TSR-011, DS-6051, PLX7486, lestatinib, darussertib, F17752, AZD6918, AZD7451, or AZ-23, or a pharmaceutically acceptable salt thereof. In some embodiments, the pharmaceutical agent is enretinib, or a pharmaceutically acceptable salt thereof. In some embodiments, the gastrointestinal NET is small intestine, large intestine, pancreas, appendix, stomach, rectum, or NET of unknown primary origin. In some embodiments, the primary NET has been transferred to a secondary tissue. In some embodiments, the secondary tissue is lymph nodes, mesentery, liver, bone, lung, or brain.

Also disclosed herein, in some embodiments, are methods of treating a gastrointestinal neuroendocrine tumor (NET) in a subject in need thereof, comprising: (a) obtaining a sample of NET genetic material from an individual; (b) determining whether NET tumors comprise an NTRK translocation or gene fusion; and (c) administering to the subject a therapeutically effective amount of an agent that inhibits a tropomyosin-receptor-kinase (Trk) protein. In some embodiments, the sample of NET genetic material is total nucleic acid obtained from a Formalin Fixed Paraffin Embedded (FFPE) tumor biopsy sample. In some embodiments, the methods disclosed herein further comprise sequencing NET genetic material to determine whether the tumor sample comprises an NTRK translocation or gene fusion. In some embodiments, the NET genetic material is sequenced by whole genome DNA sequencing, whole exome sequencing, targeted DNA sequencing, targeted RNA sequencing, or whole transcriptome RNA sequencing. In some embodiments, the methods disclosed herein further comprise amplifying NET genetic material using NTRK1, NTRK2, or NTRK 3-specific primers prior to determining whether NET tumors comprise an NTRK translocation or gene fusion. In some embodiments, the methods disclosed herein further comprise detecting an amount of NTRK expression or a level of Trk protein in a NET tumor sample, wherein an increase in the level of NTRK expression or the level of Trk protein is indicative of an NTRK translocation or gene fusion.

Is incorporated by reference

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Drawings

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

FIGS. 1A-B illustrate FDG-PET imaging of a patient. Fig. 1A illustrates patient FDG-PET imaging at the visit, revealing extensive FDG uptake in known disease areas, including massive iliac lymphadenopathy with a fusogenic mass of the left pelvic side wall, multiple hypermetabolic liver lesions, and countless lesions throughout the bone. FIG. 1B illustrates FDG-PET imaging of patients after 2 cycles of emtricinib treatment, showing improved prior fusional bone disease (compared to the proximal right femur) and decreased volume and intensity of pelvic lymphadenopathy. The most intense activity observed in the pelvis after receiving emtricinib treatment was bladder elongation, with a shift to the right.

Figures 2A-B illustrate octreotide scan (octreoscan) imaging of patients. Figure 2A illustrates anterior and posterior octreotide scan imaging (anterior and posterior, planar) of a patient at the time of visit, revealing a broad distribution of disease, including extensive skeletal metastasis and massive pelvic lymphadenopathy. Figure 2B illustrates forward and backward octreotide scan imaging of a patient after 6 cycles of emtricinib treatment. Although patients still showed signs of disease in the liver, bones and pelvis, tumor burden was significantly reduced overall compared to octreotide scanning at the visit. The largest and most active foci correspond to the massive left pelvic lateral wall and the pre-sacral mass.

Fig. 3A-B illustrate enhanced axial abdominal CT of a patient. Fig. 3A illustrates the enhanced axial abdominal CT obtained after a preliminary trial of temozolomide and capecitabine chemotherapy, showing large blocks of enhanced left pelvic lateral wall and anterior sacral tuberosity. Fig. 3B illustrates the enhanced abdominal axial CT obtained after 1 cycle of emtricinib treatment, showing a dramatic change in the appearance of the left pelvic sidewall mass, appearing swollen and more voluminous, but with reduced density.

Detailed Description

Overexpression, activation, mutation or translocation of the tropomyosin receptor kinase (Trk) family members TrkA, TrkB and TrkC has been reported in many different types of cancer, including ovarian, colorectal, melanoma and lung cancer. Trk inhibitors are effective in inhibiting both tumor growth and tumor metastasis in preclinical models of cancer. Neuroendocrine tumor (NET) is a rare, slow-growing form of cancer, caused by neuroendocrine cells distributed throughout the body. NET is an overall heterogeneous group of neoplasias, the characteristics of which may vary from tissue of origin. To date, no clear carcinogenic factors or signs of molecular parthenogenesis have been found for the vast majority of NET.

Disclosed herein, in some embodiments, are methods of treating a neuroendocrine tumor (NET) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an agent that inhibits a tropomyosin-receptor-kinase (Trk) protein, wherein the NET is associated with a Trk protein that undergoes genetic translocation or a Trk protein that is an NTRK gene fusion protein. Also disclosed herein, in some embodiments, are methods of treating a neuroendocrine tumor (NET) in a subject in need thereof, comprising: (a) obtaining a sample of NET genetic material from an individual; (b) determining whether NET tumors comprise an NTRK translocation or gene fusion; and (c) administering to the subject a therapeutically effective amount of an agent that inhibits a tropomyosin-receptor-kinase (Trk) protein.

Certain terms

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any subject matter claimed. In this application, the use of the singular includes the plural unless the context clearly dictates otherwise. For example, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Further, the use of "or" means "and/or" unless the context clearly dictates otherwise. The use of the term "including" as well as other forms, such as "comprises" and "comprising," is not intended to be limiting with respect to only the listed items. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

As used herein, the term "effective amount" or "therapeutically effective amount" refers to a sufficient amount of an agent or compound (e.g., a Trk inhibitor as described herein) that will alleviate one or more symptoms of the disease or condition being treated to some extent. The result can be a reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. For example, the term "effective amount" or "therapeutically effective amount" includes a prophylactically effective amount.

As used herein, the term "about" or "approximately" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which error range will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within one or more than one standard deviation, as practiced in the art. Alternatively, "about" may refer to a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may refer to within an order of magnitude, within 5-fold, more preferably within 2-fold of the value.

The terms "subject," "individual," and "patient" are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. In some embodiments, mammals include, but are not limited to, rats, monkeys, humans, farm animals, sport animals, and pets. In some embodiments, tissues, cells, and progeny thereof, of a biological entity obtained in vivo or cultured in vitro are included. As used herein, neither of these terms require supervision by a medical professional.

Neuroendocrine tumor (NET)

Neuroendocrine cells are highly specialized nerve-like cells that release hormones in response to neural or chemical signals. The neuroendocrine system is composed of a network of neuroendocrine cells arranged in a single organ and distributed widely throughout the body as discrete elements. For example, neuroendocrine cells are distributed throughout the gastrointestinal tract and secrete hormones that regulate intestinal motility (e.g., 5-hydroxytryptamine). On the other hand, the pituitary is a neuroendocrine gland that secretes hormones (e.g., growth hormones) that regulate a variety of physiological processes, including growth, blood pressure, and metabolism.

Neuroendocrine tumor (NET) is a rare, typically slow-growing tumor composed of neuroendocrine cells. NET originates in multiple organs and is the most common type of small bowel tumor. In some cases, NET is referred to as "carcinoid tumor", generally referring to NET originating from the diffuse neuroendocrine system, mainly the gastrointestinal and respiratory tracts. The most common site of gastrointestinal NET is the small intestine, most commonly found in the ileum, but NET also often originates in the rectum, colon, appendix and stomach.

The current understanding of NETs is obscured by the lack of a standardized naming, staging and ranking system. Furthermore, NET is often overlooked in differential diagnosis, as it is a rare form of cancer and often shows non-specific clinical manifestations. Furthermore, even small NET (<2cm) can be invasive and easily metastatic, presenting considerable challenges to the diagnosing clinician. Most studies on NET have focused on the most common sites, such as the pancreas and small intestine, which limits the widespread understanding of other less common forms of disease.

Some features are common to all NETs, while others are due to their organ of origin (e.g., gastrointestinal NETs tend to be genomically stable, while pancreatic NETs often exhibit chromosomal instability). With this degree of clinical heterogeneity, the molecular pathogenesis of NET remains elusive and the exact disease carcinogen remains undetermined. Although most pancreatic or gastrointestinal NETs are sporadic, many genetic diseases are risk factors for developing the disease. For example, multiple endocrine neoplasia type 1 (hereditary MEN1 mutation), neurofibromatosis type 1 (hereditary NF1 mutation), von Hippel-Lindau disease (hereditary VHL mutation), and tuberous sclerosis (hereditary TSC1 or TSC2 mutation) are all diseases that increase the likelihood of NET.

Disclosed herein, in some embodiments, are methods of treating a neuroendocrine tumor (NET) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an agent that inhibits a tropomyosin-receptor-kinase (Trk) protein, wherein the NET is associated with a Trk protein that undergoes genetic translocation or a Trk protein that is an NTRK gene fusion protein. Also disclosed herein, in some embodiments, are methods of treating a neuroendocrine tumor (NET) in a subject in need thereof, comprising: (a) obtaining a sample of NET genetic material from an individual; (b) determining whether NET tumors comprise an NTRK translocation or gene fusion; and (c) administering to the subject a therapeutically effective amount of an agent that inhibits a tropomyosin-receptor-kinase (Trk) protein.

In some embodiments, NET is foregut NET, midgut NET or hindgut NET. In some embodiments, the NET is a gastrointestinal NET. In some embodiments, the NET is a small intestine NET (SI-NET). In some embodiments, the NET is a large intestine NET. In some embodiments, NET is pancreatic NET (pnet). In some embodiments, the NET is the appendiceal NET. In some embodiments, the NET is gastric NET. In some embodiments, the NET is a rectal NET. In some embodiments, NET is bronchial NET. In some embodiments, the NET is ovarian NET. In some embodiments, NET is thyroid NET. In some embodiments, the NET is an unknown primary NET, wherein the organ of origin cannot be identified. In some embodiments, the primary NET has been transferred to a secondary tissue. In some embodiments, the secondary tissue is lymph nodes, mesentery, liver, bone, lung, or brain.

Carcinoid syndrome

Carcinoid syndrome may occur in some individuals carrying NET, especially in individuals with advanced or metastatic NET. Carcinoid syndrome is caused by the unregulated secretion of hormones into the blood by NET cells, which, depending on the type of NET, cause various signs and symptoms. The most common signs and symptoms of carcinoid syndrome include flushing of the skin, facial skin lesions, diarrhea, dyspnea, and increased heart rate. Patients with carcinoid syndrome often experience delayed diagnosis or misdiagnosis because symptoms may be mistaken for other diseases, such as Irritable Bowel Syndrome (IBS) or menopause.

In some embodiments, the individual has carcinoid syndrome. In some embodiments, the methods described herein further comprise administering a treatment for carcinoid syndrome. In some embodiments, the treatment for carcinoid syndrome is an anti-5-hydroxytryptamine agent. In some embodiments, the anti-5-hydroxytryptamine agent is a somatostatin analog. In some embodiments, the somatostatin analog is octreotide, octreotide acid, pasireotide, or lanreotide.

NTRK translocation and Gene fusion

The tropomyosin receptor kinase (Trk or Trk) family of tyrosine kinase receptors are multi-domain transmembrane proteins that play an important role in a wide range of neuronal responses including survival, differentiation, growth and regeneration. Trk receptors are abundantly expressed in the nervous system as well as in many other non-neuronal cell types and tissues, including monocytes, lung, bone and pancreatic beta cells. The Trk family has three members: TrkA, TrkB and TrkC, encoded by the NTRK1, NTRK2 and NTRK3 genes, respectively. TrkA, TrkB and TrkC are characterized as high affinity receptors for naturally occurring neurotrophins belonging to the family of protein growth factors, including Nerve Growth Factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4/5 (NT-4/5). Mature neurotrophins bind selective Trk receptors (e.g., TrkB-BDNF, TrkA-NGF, and TrkC-NT-3) with relatively high affinity, resulting in the activation of intracellular tyrosine kinase signaling cascades (e.g., SHC-RAS-MAPK, PI3K-AKT, or PLC γ -PKC) that mediate neurotrophin functions (e.g., neuronal growth and survival).

NTRK translocation and gene fusion proteins

Gene fusion, which is the process of fusing the complete or partial sequences of two or more different genes into a single chimeric gene or transcript, may be the result of a translocation, deletion, or inversion. While some gene fusion products are inert and do not cause significant phenotypic changes, other fusion proteins have been shown to have oncogenic activity. The incidence of gene fusion in cancer varies widely among different types of cancer. For example, TMPRSS2-ERG fusion is found in more than 50% of prostate cancer patients. In addition, some gene fusions, such as KIF5B-RET fusions, are found only in 1-2% of lung adenocarcinomas.

Oncogenic activity resulting from a translation or gene fusion event is typically due to deregulation of one of the genes involved (e.g., fusion of a strong promoter to a proto-oncogene), resulting in loss of function (e.g., by truncation of the tumor suppressor gene) or formation of a fusion protein with oncogenic function (e.g., resulting in structural activation of a tyrosine kinase, such as the NTRK gene described herein). A typical genetic structure for oncogenic tyrosine kinase gene fusion is the fusion of a catalytic kinase domain (the intracellular C-terminal region of a receptor tyrosine kinase) to an N-terminal region derived from another gene. The resulting novel oncogenes are aberrantly expressed from exogenous promoters and may result in structural activation of the kinase domain (e.g., via fusion protein dimerization mediated by the N-terminal domain).

The NTRK gene family is a promiscuous gene fusion partner and is known to produce a variety of oncogenic translocations and fusion proteins. Disclosed herein, in some embodiments, are methods of treating a neuroendocrine tumor (NET) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an agent that inhibits a tropomyosin-receptor-kinase (Trk) protein, wherein the NET is associated with a Trk protein that undergoes genetic translocation or a Trk protein that is an NTRK gene fusion protein. Also disclosed herein, in some embodiments, are methods of treating a neuroendocrine tumor (NET) in a subject in need thereof, comprising: (a) obtaining a sample of NET genetic material from an individual; (b) determining whether NET tumors comprise an NTRK translocation or gene fusion; and (c) administering to the subject a therapeutically effective amount of an agent that inhibits a tropomyosin-receptor-kinase (Trk) protein.

In some embodiments, the NTRK translocation is a translocation (including an inversion) of at least a portion of the NTRK1, NTRK2, or NTRK3 gene to another location within the same chromosome. In some embodiments, the NTRK translocation is a translocation of at least a portion of the NTRK1, NTRK2, or NTRK3 gene to a different chromosome. In some embodiments, the NTRK translocation is a translocation of at least a portion of an NTRK1, NTRK2, or NTRK3 gene that results in an NTRK1, NTRK2, or NTRK3 gene under the transcriptional control of an exogenous promoter, thereby resulting in aberrant NTRK expression. In some embodiments, NTRK translocation results in a constitutively active NTRK1, NTRK2, or NTRK3 gene.

In some embodiments, the NTRK fusion protein (also referred to as a Trk fusion protein) results from a genetic translocation of at least a portion of an NTRK1, NTRK2, or NTRK3 gene with at least a portion of another gene. In some embodiments, the NTRK fusion protein comprises an NTRK1, NTRK2, or NTRK3 tyrosine kinase signaling domain. In some embodiments, the NTRK fusion protein is under the transcriptional control of a foreign promoter, resulting in aberrant expression of NTRK. In some embodiments, the NTRK fusion protein produces a constitutively active NTRK gene. In some embodiments, the NTRK fusion protein comprises: an N-terminal region corresponding to a protein other than a TrkA, TrkB or TrkC protein and a C-terminal region corresponding to a TrkA, TrkB or TrkC protein. In some embodiments, the C-terminal region corresponding to a TrkA, TrkB or TrkC protein has TrkA, TrkB or TrkC kinase activity. As used herein, the phrase "kinase activity" means having kinase enzyme activity as understood by those of skill in the art, and includes, for example, phosphorylation of an amino acid side chain such as serine, threonine, or tyrosine. In some embodiments, the NTRK fusion protein produces a constitutively active TrkA, TrkB, or TrkC fusion protein. In some embodiments, the constitutively active TrkA, TrkB or TrkC fusion protein comprises an N-terminal region from a protein other than TrkA, TrkB or TrkC that causes dimerization of the Trk fusion protein.

In some embodiments, the NTRK fusion protein comprises a nucleic acid sequence comprising: (a) a first region corresponding to a sequence from the MPRIP, CD74, RABGAP1L, TPM3, TPR, TFG, PPL, CHTOP, ARHGEF2, NFASC, BCAN, LMNA, TP53, QKI, NACC2, VCL, AGBL4, TRIM24, PAN3, AFAP1, SQSTM1, ETV6, BTBD1, LYN, RBPMS, RFWD2, IRF2BP2, SSBP2, C18ORF8, RNF213, TBC1, DNEKHA 6, PEAR1, MRPL24, MDM4, GRIPAP1, EPS15, DYNC2H1, CEL, EPHB2, EML4, HOMER2, TEL or FAT1 gene sequence; and (b) a second region corresponding to an NTRK1, NTRK2, or NTRK3 gene sequence.

In some embodiments, the NTRK fusion protein comprises a nucleic acid sequence comprising: (a) a first region corresponding to a portion of a sequence from MPRIP, CD74, rablap 1L, TPM3, TPR, TFG, PPL, CHTOP, ARHGEF2, NFASC, BCAN, LMNA, TP53, QKI, NACC2, VCL, AGBL4, TRIM24, PAN3, AFAP1, SQSTM1, ETV6, BTBD1, LYN, RBPMS, RFWD2, IRF2BP2, SSBP2, C18ORF8, RNF213, TBC1, dnekha 6, PEAR1, MRPL24, MDM4, grippap 1, EPS15, DYNC2H1, CEL, EPHB2, EML4, HOMER2, TEL or FAT1 gene sequences; and (b) a second region corresponding to a portion of a NTRK1, NTRK2, or NTRK3 gene sequence.

In some embodiments, the NTRK fusion protein comprises: (a) an N-terminal polypeptide region comprising a polypeptide sequence other than a TrkA, TrkB or TrkC polypeptide sequence; and (b) a C-terminal polypeptide region comprising a TrkA, TrkB or TrkC polypeptide sequence, wherein the C-terminal polypeptide region has TrkA, TrkB or TrkC kinase activity. In some embodiments, the NTRK fusion protein comprises: (a) an N-terminal polypeptide region comprising the sequence of an MPRIP, CD74, RABGAP1L, TPM3, TPR, TFG, PPL, CHTOP, ARHGEF2, NFASC, BCAN, LMNA, TP53, QKI, NACC2, VCL, AGBL4, TRIM24, PAN3, AFAP1, SQSTM1, ETV6, BTBD1, LYN, RBPMS, RFWD2, IRF2BP2, SSBP2, C18ORF8, RNF213, TBC1, DNEKHA 6, PEAR1, MRPL24, MDM4, GRIPAP1, EPS15, DYNC2H1, CEL, EPHB2, EML4, HOMER2, TEL or FAT1 polypeptide sequence; and (b) a C-terminal polypeptide region comprising a TrkA, TrkB or TrkC polypeptide sequence, wherein the C-terminal polypeptide region has TrkA, TrkB or TrkC kinase activity. In some embodiments, the NTRK fusion protein is an ETV6: NTRK fusion protein and comprises an N-terminal polypeptide region comprising an ETV6 polypeptide sequence and a C-terminal polypeptide region comprising a TrkA, TrkB, or TrkC polypeptide sequence, wherein the C-terminal polypeptide region has TrkA, TrkB, or TrkC kinase activity.

In some embodiments, the Trk fusion protein comprises: (a) an N-terminal polypeptide region comprising a polypeptide sequence other than a TrkA, TrkB or TrkC polypeptide sequence; and (b) a C-terminal polypeptide region comprising a TrkA polypeptide sequence, wherein the C-terminal polypeptide comprises TrkA kinase activity; and wherein the fusion protein is TP53-TrkA, LMNA-TrkA, CD74-TrkA, TFG-TrkA, TPM3-TrkA, NFASC-TrkA, BCAN-TrkA, MPRIP-TrkA, TPR-TrkA, RFWD2-TrkA, IRF2BP2-TrkA, SQSTM1-TrkA, SSBP2-TrkA, RABGAP1L-TrkA, C18ORF8-TrkA, RNF213-TrkA, TBC1D22A-TrkA, C200RF112-TrkA, DNER-TrkA, ARHGEF2-TrkA, CHTOP-TrkA, PPL-TrkA, PLEKHA 2-TrkA, PEAR1-TrkA, MRPL24-TrkA, MDM4-TrkA, TRRC 8-TrkA, GRAP 6-TrkA, GRLR 6-TrkA, TrkYN A, EPC 27-TrkA, EPWD 27-TrkA or EPWD-TrkA.

In some embodiments, the Trk fusion protein comprises: (a) an N-terminal polypeptide region comprising a polypeptide sequence other than a TrkA, TrkB or TrkC polypeptide sequence; and (b) a C-terminal polypeptide region comprising a TrkB polypeptide sequence, wherein the C-terminal polypeptide comprises TrkB kinase activity; and wherein the fusion protein is NACC2-TrkB, QKI-TrkB, AFAP1-TrkB, PAN3-TrkB, SQSTM1-TrkB, TRIM24-TrkB, VCL-TrkB, AGBL4-TrkB or DAB2IP-TrkB fusion protein.

In some embodiments, the Trk fusion protein comprises: (a) an N-terminal polypeptide region comprising a polypeptide sequence other than a TrkA, TrkB or TrkC polypeptide sequence; and (b) a C-terminal polypeptide region comprising a TrkC polypeptide sequence, wherein the C-terminal polypeptide comprises TrkC kinase activity; and wherein the fusion protein is an ETV6-TrkC, BTBD1-TrkC, LYN-TrkC, RBPMS-TrkC, EML4-TrkC, HOMER2-TrkC, TFG-TrkC, FAT1-TrkC, or TEL-TrkC fusion protein. In some embodiments, the Trk fusion protein is an ETV6-NTRK3(ETV6-TrkC) fusion protein.

Method of treatment

Disclosed herein, in some embodiments, are methods of treating a neuroendocrine tumor (NET) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an agent that inhibits a tropomyosin-receptor-kinase (Trk) protein, wherein the NET is associated with a Trk protein that undergoes genetic translocation or a Trk protein that is an NTRK gene fusion protein. Also disclosed herein, in some embodiments, are methods of treating a neuroendocrine tumor (NET) in a subject in need thereof, comprising: (a) obtaining a sample of NET genetic material from an individual; (b) determining whether NET tumors comprise an NTRK translocation or gene fusion; and (c) administering to the subject a therapeutically effective amount of an agent that inhibits a tropomyosin-receptor-kinase (Trk) protein.

In some embodiments, the agent that inhibits a tropomyosin-receptor-kinase (Trk) protein is enretinib. Enrofloxacin (RXDX-101/NMS-E628) is a selective inhibitor of TrkA, TrkB and TrkC receptor tyrosine kinases encoded by the NTRK1-3 genes, respectively. In addition to its activity on native Trk proteins, enrcotinib is a potent inhibitor of Trk fusion proteins produced by, for example, genetic translocation or rearrangement of the NTRK gene. Enrotinib also inhibits the ROS1 and ALK proteins and ROS1 and ALK gene fusion products. Enretinib is a substituted indazole derivative having the chemical name N- [5- (3, 5-difluorophenyl) -1 h-indazol-3-yl ] -4- (4-methylpiperazin-1-yl) -2- (tetrahydropyran-4-ylamino) benzamide. See U.S. patent 8,299,057, which is incorporated herein by reference, for a description of enrotinib and other substituted indazole derivative kinase inhibitors that can be used as medicaments according to the present disclosure.

In some embodiments, the agent that inhibits the Trk protein is RXDX-102 (NMS-P360). RXDX-102 is an orally available selective tyrosine kinase inhibitor designed as an oncogene-targeted therapeutic candidate for treating cancer patients carrying altered activation of TrkA, TrkB or TrkC proteins.

In some embodiments, the agent that inhibits the Trk protein is octreotinib (DCC-2701). Octreotide is a kinase inhibitor designed to block multiple cancer signaling mechanisms in both tumor cells and the tumor microenvironment to prevent the growth and spread of cancer. The octreotinib is a MET, TIE2, VEGFR2 and TrkA/B/C kinase inhibitor with the chemical name of N- [4- [2- (cyclopropylcarboxamido) pyridin-4-yl ] oxy-2, 5-difluorophenyl ] -N- (4-fluorophenyl) cyclopropane-1, 1-dicarboxamide. See us patent 8,637,672 for a description of erlotinib and other cyclopropyl dicarboxamide compounds and analogs that can be used as agents according to the present disclosure, which is incorporated herein by reference.

In some embodiments, the agent that inhibits the Trk protein is erlotinib (LOXO-101/ARRY-470). Erlotinib is a selective Trk kinase inhibitor, currently tested in advanced solid tumors carrying NTRK fusions, with the chemical name (S) -N- [5- [ (R) -2- (2, 5-difluorophenyl) pyrrolidin-1-yl ] pyrazolo [1,5-a ] pyrimidin-3-yl ] -3-hydroxypyrrolidine-1-carboxamide. See U.S. patent 8,513,263 for a description of erlotinib and other substituted pyrazolo [1,5-a ] pyrimidine compounds that may be used as medicaments according to the present disclosure, which is incorporated herein by reference.

In some embodiments, the agent that inhibits the Trk protein is LOXO-195. LOXO-195 is a second generation selective TRK inhibitor, exhibits potent inhibition of TRK fusion proteins, and is not affected by certain acquired resistance mutations (e.g., TRKAG595R, TrkA G667C, or TrkC G623R) that may be present in patients who have been vaccinated against lenotetinib (LOXO-101) or multi-kinase inhibitors with anti-TRK activity.

In some embodiments, the agent that inhibits the Trk protein is cetrortinib (MGCD 516). Cetroritinib is a clinical-stage, orally-administrable, potent small molecule kinase inhibitor targeting a range of closely related tyrosine kinases including RET, CBL, CHR4q12, DDR and TRK. Cetrortinib is effective in inhibiting TRK fusion protein, and has the chemical name of N- [ 3-fluoro-4- [2- [5- [ (2-methoxyethylamino) methyl ] pyridin-2-yl ] thieno [3,2-b ] pyridin-7-yloxy ] phenyl ] -N- (4-fluorophenyl) cyclopropane-1, 1-dicarboxamide. See us patent 8,404,846, which is incorporated herein by reference, for a description of cetroritinib and other compounds useful as agents according to the present disclosure.

In some embodiments, the agent that inhibits the Trk protein is cabozantinib (XL 184). Cabozantinib is an orally available small molecule kinase inhibitor against RET, MET, VEGFR-1/2/3, KIT, TRKB, FLT-3, AXL and TIE-2. Cabozantinib has been clinically approved for the treatment of patients with advanced Renal Cell Carcinoma (RCC) or progressive metastatic medullary thyroid carcinoma, under the chemical name N- (4- (6, 7-dimethoxyquinolin-4-yloxy) phenyl) -N' - (4-fluorophenyl) cyclopropane-1, 1-dicarboxamide. See U.S. patent 7,579,473, which is incorporated herein by reference, for a description of cabozantinib and other compounds that can be used as medicaments according to the present disclosure.

In some embodiments, the agent that inhibits the Trk protein is mellitinib (LY 2801653). Melotinib is an orally available small molecule kinase inhibitor that interferes with signal transduction of MET, MST1R, FLT3, AXL, MERKT, TEK, ROS1, TRKA/B/C, DDR1/2, and MKNK 1/2. Mellitinib is currently being tested as a treatment for patients with advanced solid tumors with NTRK rearrangement, with the chemical name N- (3-fluoro-4- (1-methyl-6- (1H-pyrazol-4-yl) -1H-indazol-5-yloxy) phenyl) -1- (4-fluorophenyl) -6-methyl-2-oxo-1, 2-dihydropyridine-3-carboxamide. See U.S. patent 8,030,302, which is incorporated herein by reference, for a description of mellitinib and other amidophenoxy indazole compounds that can be used as medicaments according to the present disclosure.

In some embodiments, the agent that inhibits the Trk protein is dovirtinib (TKI 258). The dovitinib is a benzimidazole-quinolinone small molecule kinase inhibitor and has potential anti-tumor activity. Dovistinib binds to FGFR, PDGFR, VEGF, cKIT, FLT3, CSFR1, Trk and RET and inhibits their phosphorylation, and is currently being clinically tested for the treatment of solid tumors and/or hematologic malignancies with NTRK1 translocation.

In some embodiments, the agent that inhibits the Trk protein is zotinib. Zoltinib is an orally available small molecule kinase inhibitor approved for the treatment of patients with locally advanced or metastatic ALK-positive non-small cell lung cancer (NSCLC). Azotinib is an inhibitor against TRKA in addition to ALK, MET and ROS1, and has been used to treat NSCLC with MPRIP-NTRK1 fusion. The chemical name of azoltinib is (R) -3- [1- (2, 6-dichloro-3-fluorophenyl) ethoxy ] -5- [1- (piperidin-4-yl) -1H-pyrazol-4-yl ] pyridin-2-amine. See U.S. patent 8,785,632 for a description of zotinib and other optically pure (aminoheteroaryl) compounds that can be used as medicaments according to the present disclosure, which is incorporated herein by reference.

In some embodiments, the agent that inhibits the Trk protein is TSR-011. TSR-011(Tesoro, Inc.) is an orally available small molecule kinase inhibitor for ALK and TRK-A/B/C. TSR-011 is currently undergoing phase I/IIa trials for patients with advanced solid tumors or lymphomas with NTRK alterations.

In some embodiments, the agent that inhibits the Trk protein is DS-6051. DS-6051(Daiichi Sankyo, Inc.) is an orally available kinase inhibitor against ROS1 and TRK. DS-6051 is being used in early clinical trials for the treatment of advanced solid malignancies carrying ROS1 or NTRK gene fusions.

In some embodiments, the agent that inhibits the Trk protein is PLX 7486. PLX7486(Plexxikon) is an orally administrable kinase inhibitor in early clinical trials for the treatment of advanced solid malignancies with activating trk (NTRK) mutations or NTRK gene fusions.

In some embodiments, the agent that inhibits the Trk protein is lestaurtinib (CEP-701). Lestaurtinib is an orally available indolocarbazole derivative kinase inhibitor against FLT3, JAK2 and TrkA/B/C.

In some embodiments, the agent that inhibits the Trk protein is dalutasertib (PHA-739358), F17752(Pierre Fabre), AZD6918(Astra Zeneca), AZD7451(Astra Zeneca), or AZ-23(CAS #: 915720-21-7).

Various pharmaceutically acceptable salts, carriers or excipients of the Trk inhibitors described above are available, and any suitable pharmaceutically acceptable salt, carrier or excipient is contemplated for use with the Trk inhibitors disclosed herein.

In some embodiments, an agent that inhibits tropomyosin-receptor-kinase (Trk) protein is used as a primary or first-line therapy. In some embodiments, the agent that inhibits the Trk protein is used as a secondary or rescue therapy. In some embodiments, an agent that inhibits the Trk protein is used as a secondary or rescue therapy, wherein the individual has a stable or progressive disease following a previous chemotherapeutic treatment regimen. A variety of chemotherapeutic agents are available and known in the art, and any suitable chemotherapy is contemplated for use with the methods disclosed herein. Exemplary chemotherapeutic treatment regimens include, but are not limited to, capecitabine, 5-fluorouracil, doxorubicin, etoposide, dacarbazine, streptozotocin, temozolomide, cisplatin, cyclophosphamide, thalidomide, or any combination thereof.

Disclosed herein, in some embodiments, are methods of treating a neuroendocrine tumor (NET) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an agent that inhibits a tropomyosin-receptor-kinase (Trk) protein, wherein the NET is associated with a Trk protein that undergoes genetic translocation or a Trk protein that is an NTRK gene fusion protein. Also disclosed herein, in some embodiments, are methods of treating a neuroendocrine tumor (NET) in a subject in need thereof, comprising: (a) obtaining a sample of NET genetic material from an individual; (b) determining whether NET tumors comprise an NTRK translocation or gene fusion; and (c) administering to the subject a therapeutically effective amount of an agent that inhibits a tropomyosin-receptor-kinase (Trk) protein.

In some embodiments, methods of identifying an NTRK translocation or gene fusion include the use of microarrays, Expressed Sequence Tag (EST) mapping, karyotyping, cytogenetic analysis, phosphotyrosine signaling screening, phosphoproteomic analysis, chromatin interaction analysis, mass spectrophotometry, tandem mass spectrophotometry analysis, quantitative PCR (qpcr), digital PCR, droplet digital PCR (e.g., rainance RainDrop Plus)^TMOr Bio-RadQX200^TMDropletDigital^TMPCR), fluorescence in situ hybridization assay (FISH), or any combination thereof. In some embodiments, the method of identifying an NTRK translocation or gene fusion comprises sequencing tumor genetic material. In some embodiments, the tumor sample is subjected to DNA sequencing, whole genome sequencing, exome sequencing, or RNA sequencing to determine whether the tumor sample comprises an NTRK translocation or gene fusion.

A variety of sequencing technologies and techniques are available, and any suitable sequencing technology is contemplated for use with the methods disclosed herein. In some embodiments, the sequencing technique is a dye terminator-based sequencing methodology (e.g., Sanger sequencing). In some embodiments, the sequencing technology is a Next Generation Sequencing (NGS) method. In some embodiments, the new generation sequencing method is pyrosequencing (e.g., Roche 454 system), sequencing by synthesis (e.g., GA/HiSeq/MiSeq/NextSeq system by Illumina), sequencing by ligation (e.g., SOLiD system by Applied Biosystem), sequencing by detection of hydrogen ions released during DNA polymerization (e.g., Ion Torrent sequencing system), single molecule sequencing (e.g., Pacific Biosciences sequencing system), or any combination thereof. In some embodiments, the genetic material is DNA or complementary DNA (cdna). In some embodiments, the cDNA is reverse transcribed from RNA. In some embodiments, sequencing comprises Whole Genome Sequencing (WGS), Whole Exome Sequencing (WES), whole genome transcriptome sequencing (RNAseq), targeted sequencing methods, or any combination thereof. In some embodiments, the specific target is enriched from isolated tumor genetic material (e.g., exome, or more preferably, NTRK-containing exons). In some embodiments, the genetic material is amplified prior to detection of the NTRK fusion by, for example, DNA or RNA sequencing.

In some embodiments, prior to sequencing (e.g., by NGS), the genetic material is broken down into a plurality of nucleic acid fragments to create a sequencing library. A variety of library preparation techniques are available, and any suitable method for creating a sequencing library is contemplated for use with the methods disclosed herein. In some embodiments, the size of the plurality of nucleic acid fragments is selected prior to sequencing. In some embodiments, the plurality of nucleic acid fragments comprises a barcode for identifying the plurality of fragments, e.g., in a multiplex reaction containing different samples. In some embodiments, the barcode is a molecular-scale barcode. In some embodiments, the barcode is a sample-grade barcode. In some embodiments, sequencing the genetic material comprises sequencing one end (single-ended) of the plurality of nucleic acid fragments. In some embodiments, sequencing the genetic material comprises sequencing both ends (paired ends or paired ends) of the plurality of nucleic acid fragments. In some embodiments, the method of identifying an NTRK translocation or gene fusion comprises paired-end RNA sequencing (PE-RNAseq).

In some embodiments, the high throughput sequencing method is coupled with a computational tool for identifying NTRK translocations or gene fusions. A variety of computing tools are available, and any suitable computing tool is contemplated for use with the methods disclosed herein. Exemplary computing tools include, but are not limited to, Fusion MetaCaller, INTEGRATE, IDP-Fusion, JAFFA, TRUP, ChildDecode, Fusion catcher, PRADA, EBARDenovo, Fusion Q, iFUSE, SOAPFUSE, SOAPfusion, Bellerophontes, Breakfusion, ElDorado, EricScript, Fusion Analyzer, Fusion Finder, Lifesscope, nuse, ChimeraScan, Comrad, defuette, Fusion Hunter, Fusion Map, ShortFuse, SnowShoes-FTD, TopH-Fusion, Fusion Seq, or any combination thereof.

In some embodiments, the gene fusion is verified using Polymerase Chain Reaction (PCR), for example by PCR, RT-PCR, qPCR, or any combination thereof.

Combination therapy

Disclosed herein, in some embodiments, are methods of treating a neuroendocrine tumor (NET) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an agent that inhibits a tropomyosin-receptor-kinase (Trk) protein, wherein the NET is associated with a Trk protein that undergoes genetic translocation or a Trk protein that is an NTRK gene fusion protein. Also disclosed herein, in some embodiments, are methods of treating a neuroendocrine tumor (NET) in a subject in need thereof, comprising: (a) obtaining a sample of NET genetic material from an individual; (b) determining whether NET tumors comprise an NTRK translocation or gene fusion; and (c) administering to the subject a therapeutically effective amount of an agent that inhibits a tropomyosin-receptor-kinase (Trk) protein.

In some embodiments, the method further comprises administering additional therapy to the individual. In some embodiments, the additional therapy is a second agent that inhibits a Trk protein (e.g., a Trk inhibitor disclosed herein). In some embodiments, the NET has primary resistance to an agent that inhibits the Trk protein. In some embodiments, the NET develops acquired (secondary) resistance to an agent that inhibits the Trk protein. In some embodiments, the NET having acquired resistance to an agent that inhibits the Trk protein comprises a mutation in a kinase domain of the Trk protein. In some embodiments, the mutation in the kinase domain is a TrkAG595R or G667C mutation. In some embodiments, the mutation in the kinase domain is located in the kinase domain of the TrkB protein and corresponds to a TrkA G595R or G667C mutation. In some embodiments, the mutation in the kinase domain is located in the kinase domain of the TrkC protein and corresponds to a TrkC G595R or G667C mutation. The amino acid sequences of human TrkA, TrkB and TrkC may be obtained from various suitable databases (e.g., UniprotKB database or GenBank), and any suitable alignment program (e.g., BLAST, muccle tool, etc.) may be used to align the kinase domains of TrkA, TrkB and TrkC. In some embodiments, NET having primary resistance to an agent that inhibits the Trk protein is administered a second agent that inhibits the Trk protein, wherein the NET is non-resistant to the second agent. In some embodiments, NET having acquired resistance to an agent that inhibits the Trk protein is administered a second agent that inhibits the Trk protein, wherein NET is not resistant to the second agent.

In some embodiments, the method further comprises administering additional therapy to the individual, wherein the additional therapy comprises administering a PI3K/Akt/mTOR pathway inhibitor, a TGF- β pathway inhibitor, a cell cycle inhibitor, a somatostatin analog, an interferon, or an angiogenesis inhibitor. In some embodiments, the interferon is a type I interferon. In some embodiments, the type I interferon is IFN- α.

In some embodiments, the somatostatin analog is octreotide, octreotide acid, pasireotide, or lanreotide. In some embodiments, the somatostatin analog is radiolabeled. In some embodiments, the radiolabeled somatostatin analog is [ DOTA ]⁰,Tyr³]Octreotide acid (lutetium oxo octreotide). In some embodiments, the additional therapy is everolimus (mTor inhibitor), temsirolimus (mTor inhibitor), bevacizumab (VEGF/angiogenesis inhibitor), sunitinib, or sorafenib (VEGF/MAPK inhibitor). In some embodiments, the additional therapy comprises surgery, chemotherapy, or radiation therapy.