Mechanisms for mTORC1 activation and synergistic induction of apoptosis by ruxolitinib and BH3 mimetics or autophagy inhibitors in JAK2-V617F-expressing leukemic cells including newly established PVTL-2

Abstract

The activated JAK2-V617F mutant is very frequently found in myeloproliferative neoplasms (MPNs), and its inhibitor ruxolitinib has been in clinical use, albeit with limited efficacies. Here, we examine the signaling mechanisms from JAK2-V617F and responses to ruxolitinib in JAK2-V617F-positive leukemic cell lines, including PVTL-2, newly established from a patient with post-MPN secondary acute myeloid leukemia, and the widely used model cell line HEL. We have found that ruxolitinib downregulated the mTORC1/S6K/4EBP1 pathway at least partly through inhibition of the STAT5/Pim-2 pathway with concomitant downregulation of c-Myc, MCL-1, and BCL-xL as well as induction of autophagy in these cells. Ruxolitinib very efficiently inhibited proliferation but only modestly induced apoptosis. However, inhibition of BCL-xL/BCL-2 by the BH3 mimetics ABT-737 and navitoclax or BCL-xL by A-1331852 induced caspase-dependent apoptosis involving activation of Bak and Bax synergistically with ruxolitinib in HEL cells. On the other hand, the putative pan-BH3 mimetic obatoclax as well as chloroquine and bafilomycin A1 inhibited autophagy at its late stage and induced apoptosis in PVTL-2 cells synergistically with ruxolitinib. The present study suggests that autophagy as well as the anti-apoptotic BCL-2 family members, regulated at least partly by the mTORC1 pathway downstream of STAT5/Pim-2, protects JAK2-V617F-positive leukemic cells from ruxolitinib-induced apoptosis depending on cell types and may contribute to development of new strategies against JAK2-V617F-positive neoplasms.

The potential of JAK/STAT pathway inhibition by ruxolitinib in the treatment of COVID-19

“…Reference Effects of ruxolitinib Reference CBL Downregulated after treatment in severe cases (PBMC) [14] Inhibits CBL mutant Pluripotent Stem Cells [90] CXCL10 Upregulated in COVID-19 patients PBMC (compared to normal PBMC) and elevated blood level in severe patients [32,49,52] Downregulates the expression level in macrophages [91] IL10 Upregulated in COVID-19 patients PBMC (compared to normal PBMC)and elevated blood level in severe patients [10,11,49,50,53,14,15,16,18,19,24,27,31] Decreases secretion in macrophages [92] IL18 Upregulated COVID-19 patients PBMC (compared to normal PBMC) [49] Downregulates IL18 expression levels in lymphoblasts [69] IL2 Elevated blood level in severe patients [31] Reduces IL2 levels in T cells [73] IL2RB Downregulated after treatment in severe cases (PBMC) [14] Inhibits JAK/STAT pathway activating IL2RB mutant Ba/F3 cells [93] MCL1 Downregulated after treatment in severe cases (PBMC) [14] Downregulates expression level in lymphoblasts [94]…”

The pan-PIM inhibitor INCB053914 displays potent synergy in combination with ruxolitinib in models of MPN

“…Targeting mTOR has also demonstrated positive anti-MPN effects as a single agent and in combination with JAK2 inhibition. 45,[65][66][67][68][69] The potency of the combination of INCB053914 and ruxolitinib was also manifested in in vivo MPN models driven by aberrant JAK2 signaling. The combination of INCB053914 and ruxolitinib antagonized the growth of SET2 cell xenograft tumors at doses of each drug that had little to no effect as monotherapies ( Figure 6A).…”

Curcumin induces apoptosis in JAK2‐mutated cells by the inhibition of JAK2/STAT and mTORC1 pathways

“…Ishida et al demonstrated that HEL cells are only partially dependent on JAK2 V617F for survival and this may explain the very limited effect of ruxolitinib and its inefficiency to eradicate JAK2‐mutated clone in MPNs patients . Interestingly, in this study, we observed that curcumin affects both proliferation and survival of HEL cells, suggesting that its role in MPNs could be more effective in blocking neoplastic cells respect to the common JAK2 inhibitors, such as ruxolitinib.…”