The SMAC mimetic LCL-161 selectively targets JAK2V617F mutant cells

Abstract

Background: Evasion from programmed cell death is a hallmark of cancer and can be achieved in cancer cells by overexpression of inhibitor of apoptosis proteins (IAPs). Second mitochondria-derived activator of caspases (SMAC) directly bind to IAPs and promote apoptosis; thus, SMAC mimetics have been investigated in a variety of cancer types. particularly in diseases with high inflammation and NFĸB activation. Given that elevated TNFα levels and NFĸB activation is a characteristic feature of myeloproliferative neoplasms (MPN), we investigated the effect of the SMAC mimetic LCL-161 on MPN cell survival in vitro and disease development in vivo.

Methods: To investigate the effect of the SMAC mimetic LCL-161 in vitro, we utilized murine and human cell lines to perform cell viability assays as well as primary bone marrow from mice or humans with JAK2^V617F-driven MPN to interrogate myeloid colony formation. To elucidate the effect of the SMAC mimetic LCL-161 in vivo, we treated a JAK2^V617F-driven mouse model of MPN with LCL-161 then assessed blood counts, splenomegaly, and myelofibrosis.

Results: We found that JAK2^V617F-mutated cells are hypersensitive to the SMAC mimetic LCL-161 in the absence of exogenous TNFα. JAK2 kinase activity and NFĸB activation is required for JAK2^V617F-mediated sensitivity to LCL-161, as JAK or NFĸB inhibitors diminished the differential sensitivity of JAK2^V617F mutant cells to IAP inhibition. Finally, LCL-161 reduces splenomegaly and may reduce fibrosis in a mouse model of JAK2^V617F-driven MPN.

Conclusion: LCL-161 may be therapeutically useful in MPN, in particular when exogenous TNFα signaling is blocked. NFĸB activation is a characteristic feature of JAK2^V617F mutant cells and this sensitizes them to SMAC mimetic induced killing even in the absence of TNFα. However, when exogenous TNFα is added, NFĸB is activated in both mutant and wild-type cells, abolishing the differential sensitivity. Moreover, JAK kinase activity is required for the differential sensitivity of JAK2^V617F mutant cells, suggesting that the addition of JAK2 inhibitors to SMAC mimetics would detract from the ability of SMAC mimetics to selectively target JAK2^V617F mutant cells. Instead, combination therapy with other agents that reduce inflammatory cytokines but preserve JAK2 signaling in mutant cells may be a more beneficial combination therapy in MPN.

Keywords: Myeloproliferative neoplasm; SMAC mimetic; TNFα.

Fig. 1

JAK2^V617F mutant cells lines are more sensitive to killing by LCL-161 under certain circumstances. a–c. L929 cells expressing JAK2^WT, JAK2^V617F or empty vector were incubated with increasing concentrations of a LCL-161 alone, b LCL-161 with the addition of 0.25 ng/ml mTNFα, c LCL-161 with the addition of 400 ng/ml mTNFa neutralizing antibody for 48 h and then analyzed with a resazurin based viability assay. ****P < 0.0001, 2way ANOVA. d Western blot of L929 cell lines harvested 24 h after exposure to combinations of LCL-161 and mTNFα. e–g HEL and K562 cells were incubated with e LCL-161 alone, f LCL-161 + 1 ng/ml hTNFα, or g LCL-161 + 10 ng/ml hTNFα for 48 h. Apoptosis was measured with Annexin V and PI staining. **P < 0.01, ****P < 0.0001 unpaired t test

Fig. 2

JAK inhibitors rescue hypersensitivity of JAK2^V617F mutant cells to LCL-161. L929 cells expressing JAK2^WT, JAK2^V617F or empty vector were incubated with increasing concentrations of a LCL-161 alone, b LCL-161 with the addition of 1 µM ruxolitinib, or c LCL-161 with the addition of 1 µM pacritinib. After 48 h in culture a resazurin-based cell viability assay was performed. ****P < 0.0001, 2way ANOVA

Fig. 3

LCL-161 preferentially decreases MPN colony formation. Methylcellulose colony formation of a, b whole bone marrow from JAK2^V617F or wild-type mice and c, d peripheral blood mononuclear cells from JAK2^V617F mutated MPN patients or normal controls with increasing concentrations of LCL-161 alone (a, c) or LCL-161 + 0.25 ng/ml TNFα (b, d). *P < 0.05, ***P < 0.001, unpaired t test

Fig. 4

Impact of LCL-161 treatment in a transduction–transplantation model of JAK2V617F mutated MPN. a Percentage of GFP+ (empty vector or JAK2^V617F) cells in peripheral blood. b White blood cell (WBC), c hematocrit (HCT), d platelet (PLT) and e hemoglobin (HGB) counts in wild-type (empty vector) or JAK2^V617F mice treated with LCL-161. f Spleen weights in JAK2^V617F mice, P < 0.05, unpaired t test. g Percentage of GFP+ (JAK2^V617F) cells in the spleen and bone marrow. h Snook’s reticulin staining of paraffin-embedded bone marrows in wildtype (empty vector) and JAK2^V617F mice treated with LCL-161. n = 3–4 mice per group