In this study, we show the efficacy and tolerability of the pan HDACi pracinostat in various in vitro
and in vivo
models of AML, and show synergistic effects at multiple levels in combination with the JAK2/FLT-3 inhibitor pacritinib in both the in vitro
and in vivo
setting. We also explored the possible mechanisms for these synergistic anti-tumor effects. Previously reported synergistic effects of an HDACi combined with a JAK2 inhibitor was attributed to impaired chaperone function of heat shock protein 90 by the HDACi, promoting proteasomal degradation and depleting total JAK2 levels.21
FLT3 is another heat shock protein 90 client protein, where mutant forms such as FLT3-ITD are more dependent on the chaperone association than their wt counterpart.39
In addition, earlier studies have shown that HDAC inhibition depleted mRNA levels of JAK2V617F
Therefore, not surprisingly, we have demonstrated that pracinostat not only reduced JAK2/STAT5 protein levels in cells bearing a JAK2 mutation, but also FLT3/STAT5 levels in cells with a FLT-3 mutations (as seen in ). Recent studies with the HDACi trichostatin A demonstrate that JAK2/STAT3 signaling was decreased by upregulation of the expression of suppressors of cytokine signaling (SOCS) 1
A possible direct effect of the HDAC inhibition on the phosphorylation of JAK2, STAT5 and FLT3 was not investigated in this study.
SB939 showed potent inhibition predominantly in JAK2V617F- or FLT3-ITD-harboring cell lines (). The cell line with the lowest IC50
was HL-60, which carries an N-RAS
mutation. HDACi have been shown to block Ras-dependent signaling and growth transformation.41
Surprisingly, in HEL92.1.7 and MOLM-13 cells, the pracinostat IC50
on proliferation is lower than the IC50
on inhibition of JAK2 or FLT3-ITD protein levels, respectively. This discrepancy might be a result of modulation of other genes besides JAK2V617F
and FLT3-ITD by HDAC inhibition.
Pacritinib is an equipotent inhibitor of JAK2 and FLT3, which is effective in reducing JAK2/STAT5 and FLT-3 JAK2 signaling in JAK2 and FLT3 mutant cells, respectively.33
The combination of pracinostat and pacritinib led to synergistic effects with a complete inhibition of downstream STAT5 signaling, an increased efficacy on cell proliferation and the induction of apoptosis. In vitro
combination studies in different cell lines with either wt or mutant JAK2 or FLT3 also demonstrated synergy, mostly in cells that carried the mutant protein. One exception was the F36-P cell line. The growth of this cell line depends on exogenously added granulocyte macrophage colony-stimulating factor,42
which signals exclusively via JAK2, making it a JAK2 wt-dependent cell line. This indicates that synergy between a JAK2 inhibitor and an HDACi might also work in cells that are fully dependent on JAK2 (wt) signaling. Consistent with this, similar in vitro
synergy was observed in the JAK2 wt SET-2 cells and F36-P cells but not in FLT3 mutant cell lines with the specific pan-JAK inhibitor ruxolitinib in combination with pracinostat (data not shown).
LMO2 is a transcription factor involved in normal hematopoiesis, but also leukemogenesis that is overexpressed in many AML cells.43
Interestingly, LMO2 levels were downregulated synergistically in MOLM-13 cells with pacritinib and pracinostat, and may be a result of another synergistic interaction between JAK2 and HDAC. Dawson et al.43
demonstrate that JAK2 inhibition leads to lower levels of histone H3 Y41 phosphorylation on the promoter of LMO2, whereas increasing the binding of heterochromatin protein 1α at the same site, resulting in lower expression of LMO2. JAK2 may have an epigenetic role in the nucleus to influence the status of H3 acetylation. It has been demonstrated previously that phosphorylation of H3 (on S10) leads to increased efficiency of a subsequent H3 acetylation, resulting in synergistic modifications of gene expression.44
Pacritinib, as well as targeting JAK2, is a potent FLT3 inhibitor. Our group has recently discovered that treatment of FLT3-ITD cells with FLT3 inhibitors lacking JAK2 activity (e.g., ABT-869, VX-680 or sunitinib), leads to an upregulation of JAK2 activity, causing secondary resistance.33
Therefore, although combinations of FLT3 inhibitors and HDACi have been described to show synergy in vitro
this combination without the additional JAK2 inhibition could lead to resistance after chronic dosing and not show enhanced efficacy in the in vivo
setting. This may explain why none of the studies showing in vitro
synergy reported any in vivo
synergy data. Pacritinib as a dual JAK2/FLT-3 inhibitor is therefore ideally suited for a combination with an HDACi and superior to an inhibitor that only affects FLT3 kinase without targeting other JAK family kinases.
Although the combination of pracinostat and pacritinib showed synergy in vitro
, the synergy was greater in the in vivo
setting in both AML models tested. This indicates that there are additional synergistic mechanisms that are only working in the whole animal setting. One example is the synergistic effects observed on metastases. In AML patients, leukemia cutis and extramedullary involvement of organs such as the lungs are common. Respiratory distress syndrome secondary to lung involvement causes a significant percentage of the morbidity/mortality associated with AML.45
Therefore, the synergy observed in reducing metastatic sites in the animal model is certainly of great relevance for AML patients. Interestingly, significantly higher plasma levels of MCP-1 were measured in untreated AML patients with extramedullary sites involved than in those with complete remission.46
This highlights the potential therapeutic benefit with our observation that pracinostat and pacritinib synergistically decrease MCP-1 plasma levels as well as metastatic occurrences. In both models tested, chronic treatment with one drug alone led to the increase of a signaling pathway. Pacritinib in the Molm-13 model or pracinostat (Set-2 model) led to increased FLT3 or pSTAT5 levels, respectively, whereas the combination treatment in both studies was most efficiently suppressing the signaling, indicating that a combination treatment can overcome treatment-induced resistance.
Effects of tumor-induced elevations of cytokine and chemokine levels may be another mechanism for the synergy observed with pracinostat and pacritinib. HDACi as well as JAK2 inhibitors have been described to affect the production of various growth factors and cytokines,35, 36, 37
thereby influencing tumor growth. Manshouri et al.38
recently showed that resistance to JAK2-inhibitor treatment of MPNs is mediated by cytokines produced by the bone marrow stroma. Distinctly high levels of IL-6, FGF4 and CXCL10/IP-10 were detected in co-cultures of stromal cells and SET-2 cells, mediating resistance to the JAK2 inhibitor antiprimod. Although SCID beige mice that lack B-cells, T-cells, as well as natural killer cells, were used for the SET-2 AML model in our studies, high circulating levels IL-6, IP-10, KC and MCP-1 were measured in the tumor-bearing mice without drug treatment. Treatment with pacritinib or pracinostat as single agents led to the normalization of IL-6, IP-10, KC and MIP-1α, and a synergistic normalization of MCP-1 levels was observed with the combination treatment.
Taken together, our studies demonstrated the synergistic efficacy of a combination of pracinostat and pacritinib in in vitro and in vivo models of AML and offer mechanistic insights for this synergy. These data provide a scientific rationale for the combination of pracinostat and pacritinib for advanced acute leukemia, which warrants further exploration in a clinical trial.