Combination of PIM and JAK2 inhibitors synergistically suppresses cell proliferation and overcomes drug resistance of myeloproliferative neoplasms

Abstract

Inhibitors of JAK2 kinase are emerging as an important treatment modality for myeloproliferative neoplasms (MPN). However, similar to other kinase inhibitors, resistance to JAK2 inhibitors may eventually emerge through a variety of mechanisms. Effective drug combination is one way to enhance therapeutic efficacy and combat resistance against JAK2 inhibitors. To identify potential combination partners for JAK2 compounds in MPN cell lines, we performed pooled shRNA screen targeting 5,000 genes in the presence or absence of JAK2 blockade. One of the top hits identified was MYC, an oncogenic transcription factor that is difficult to inhibit directly, but could be targeted by modulation of upstream regulatory elements such as kinases. We demonstrate herein that PIM kinase inhibitors efficiently suppress MYC protein levels in MPN cell lines. Overexpression of MYC restores the viability of PIM inhibitor-treated cells, revealing causal relationship between MYC down-regulation and cell growth inhibition by PIM compounds. Combination of various PIM inhibitors with a JAK2 inhibitor results in significant synergistic growth inhibition of multiple MPN cancer cell lines and induction of apoptosis. Mechanistic studies revealed strong downregulation of phosphorylated forms of S6 and 4EBP1 by JAK2/PIM inhibitor combination treatment. Finally, such combination was effective in eradicating in vitro JAK2 inhibitor-resistant MPN clones, where MYC is consistently up-regulated. These findings demonstrate that simultaneous suppression of JAK2 and PIM kinase activity by small molecule inhibitors is more effective than either agent alone in suppressing MPN cell growth. Our data suggest that JAK2 and PIM combination might warrant further investigation for the treatment of JAK2-driven hematologic malignancies.

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

“…This includes the pan-PIM inhibitor AZD1208 and other structurally diverse PIM inhibitors. 36,37 Recently, we reported respectively. Note that the vinculin blot in panel A and Figure 3B are the same because these were the same drug-treated cells and subsequent lysates analyzed in each figure.…”

“…Accordingly, PIM inhibitors have been assessed as potential therapeutic targets for MPNs. [36][37][38][39] Here, we report the impressive efficacy of a recently described pan-PIM inhibitor INCB053914 40 in combination with the JAK1/2 inhibitor ruxolitinib (approved by the US Food and Drug Administration) in preclinical MPN animal and human cell models.…”

“…Exposure of cells to PIM inhibitors increases PIM protein levels 36,37,40 and impairs phosphorylation of the PIM substrate PRAS40 at threonine 246. 10,19,44 INCB053914 treatment of MPN model cells triggered increases in PIM1, PIM2, and PIM3 protein levels (supplemental Figure 2A) as well as inhibition of pPRAS40(T246) (supplemental Figure 2B), indicating on-target activity of INCB053914.…”

“…This is consistent with the ability of PIM to promote mTORC1 signaling by phosphorylating PRAS40, which antagonizes the ability of PRAS40 to inhibit mTORC1 10,11 and is consistent with the effects of other PIM inhibitors. 36,37,44,61 Because mTORC1 regulates a large number of cellular pathways, including metabolism and protein synthesis, 15 the cellular effects of the combination of ruxolitinib and INCB053914 is likely the result of the combination of altered regulation of numerous critical cellular processes. Targeting mTOR has also demonstrated positive anti-MPN effects as a single agent and in combination with JAK2 inhibition.…”

Novel Therapies for Myelofibrosis

“…Two family members, PIM1 and PIM2, have been found to be upregulated in MPN (100). PIM inhibitors have shown preclinical synergy with JAK inhibitors, as well as the ability to overcome JAK inhibitor resistance in MPN cell lines (101, 102). A phase 1b study of ruxolitinib plus PIM inhibitor PIM447, or ruxolitinib plus CDK4/6 inhibitor ribocicilib (LEE011), or the combination of all three is underway in several non-U.S. countries (NCT02370706).…”