HSP90 inhibition is efficient against human CRLF2 rearranged B ALL in vivo To extend our findings towards the in vivo therapy of human B-ALL, we established key B-ALL xenografts from CRLF2-rearranged, patient-derived bone marrow samples in NOD. Cg-Prkdcscid Il2rgtm1Wjl SzJ mice. Patient sample 412 harbors a CRLF2 IGH translocation along with a JAK2 R683S mutation. Patient sample 537 harbors a P2RY8 CRLF2 rearrangement and lacks a somatic mutation inside the recognized elements of CRLF2 signaling, according to transcriptome and exome sequencing. To stringently assay established illness in vivo, we sacrificed sentinel animals weekly just after transplantation to assess engraftment. Once bone marrow leukemia burden exceeded 30%, we initiated remedy with 50 mg kg BVB808 twice everyday by oral gavage, 50 mg kg AUY922 thrice weekly i. v, BVB808 AUY922, or car.
The dose of BVB808 selelck kinase inhibitor was selected according to the demonstrated activity at this dose in Jak2V617F driven MPNs and prior studies that demonstrated weight loss at higher doses. Immediately after 5 d of remedy, we sacrificed animals to assess pharmacodynamic Spleens from mice treated with vehicle or BVB808 had almost total effacement by B-ALL, whereas AUY922 or BVB808 AUY922 treatment resulted in visible islands of hematopoiesis. Depending on immunohistochemistry, mice receiving AUY922 or BVB808 AUY922, but not BVB808 or automobile, had almost full loss of pSTAT5 and up-regulation of HSP70. Immunoblotting of spleens from treated mice demonstrated comparable findings to these observed right after treatment of MUTZ5 and MHH- CALL4, particularly, reductions in pSTAT5, pJAK2, and total JAK2 in AUY922- or BVB808 AUY922- treated mice. In contrast, remedy with single- agent BVB808 only modestly suppressed pSTAT5. As noted in MHH-CALL4 cells, therapy with either BVB808 or AUY922 decreased pSTAT1.
We performed transcriptional profiling on bone marrow from mice following five d of therapy. Unsupervised hierarchical clustering demonstrated the exact same pattern of clus- tering observed after therapy of B-ALL cell lines. Specifically, mice treated with AUY922 or BVB808 AUY922 clustered with each other, whereas vehicle- and BVB808-treated mice clustered together, indicating the dominant impact of HSP90 selleck chemical inhibition. Remedy with either BVB808 or AUY922 prolonged general survival compared with car. Treatment with AUY922 additional pro- longed general survival compared with BVB808, whereas the combination of BVB808 and AUY922 had no further advantage compared with AUY922 alone. DISCUSSION In this study, we describe point mutations close to the ATP- binding region from the JAK2 kinase domain that confer resistance to a broad panel of enzymatic JAK inhibitors. All 3 mutations are in regions homologous to imatinib resis- tance hotspots in ABL1 and market multiagent resistance inside the context of Jak2 V617F or JAK2 R683G.