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Promising preclinical data on combined JAK, PIM, and CDK4/6 inhibition in MPN

May 13, 2021
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Ruxolitinib, a selective inhibitor of Janus kinase 1/2 (JAK1/2), has proven to be beneficial to patients with Philadelphia-chromosome negative myeloproliferative neoplasms (MPN), in reducing spleen size and improving constitutional symptoms. However, 51% of patients discontinue therapy due to suboptimal response, disease progression, or anemia within 3 years after start. For this reason, there is a clinical need for treatment strategies to enhance tolerability and efficacy of ruxolitinib.

Somatic mutations in the JAK2, MPL, or CARL genes lead to the aberrant activation of JAK-STAT signaling, which is considered a hallmark in MPN. This in turn, results in the dysregulation of key downstream pathways, ultimately increasing the expression of cell cycle mediators such as CDC25A and the PIM kinases; thus, contributing to MPN pathogenesis.

A recent preclinical study by Raajit K. Rampal and colleagues investigated the therapeutic efficacy of a triple combination of ruxolitinib, the PIM kinase inhibitor PIM447, and the CDK4/6 inhibitor LEE011 in patients with MPN.1 We hereby provide a summary of the key results.

Study design

Murine models

For the UKE-1 model:

  • JAK2V617F knock-in and MPLW515L-mutant mice were used.
  • 14 days following injection with 10 million viable bone marrow cells, mice were randomized to receive:
    • Vehicle
    • 60 mg/kg ruxolitinib twice daily, or
    • combined 60 mg/kg ruxolitinib twice daily, 12.5 mg/kg PIM447 daily, and 37.5 mg/kg LEE011 daily.
  • Nonlethal bleeds were performed 14 days after transplantation and every 14−30 days thereafter to assess disease severity.

JAK2 mutant cell lines

BaF3-JAK2V617-mutant UKE-1 cell lines were grown.

  • 24 hours after plating, UKE-1 cells were treated with 300 nM ruxolitinib, 300 nM PIM447, and 1,000 nM LEE011, either individually or in combination (doublet and triplet).
  • Phosphorylated and total proteins were measured and evaluated for apoptosis and cell cycle assays.

Primary MPN patient samples

  • Peripheral blood mononuclear cells and CD34+ cells were isolated and cultured with vehicle, ruxolitinib or triple therapy for 48 hours.
  • Cells were then harvested and used for Western Blot analysis.

Results1

In vitro results

JAK2 mutant cell lines (in a JAK2V617F-mutant UKE-1 leukemia xenograft model)

    • While doublet combinations (ruxolitinib + LEE011, or LEE011 + PIM447) could induce tumor regression, the triple combination (ruxolitinib + LEE011 + PIM447) was associated with complete tumor regression.
    • Triple combination resulted in expected on-target pharmacodynamic effects:
      • Reductions in pSTAT-5 levels were similar with ruxolitinib alone and triple combination.
      • A proportion of cells in S-phase significantly decreased with the triple combination versus ruxolitinib exposed cells (p < 0.0001), and vs those treated with a doublet combination of ruxolitinib and PIM447 (p < 0.001) or a doublet combination of ruxolitinib and LEE011 (p < 0.01).
      • Apoptosis (early and late) significantly increased with triple combination vs ruxolitinib (p < 0.0001), the ruxolitinib and PIM447 (p < 0.001) or ruxolitinib and LEE011 (p < 0.01) doublets.

    In vivo results

        In murine models of JAK2V617F and MPLW515L-mutant MPN, triple combination therapy (vs ruxolitinib alone if not stated):

        • Eliminated disease-initiating capacity of treated bone marrow vs vehicle.
        • Reduced tumor burden by 238-fold (p < 0.001) vs 3.4-fold with ruxolitinib alone.
        • Significantly reduced spleen size (p < 0.0001).
        • Significantly reduced mutant JAK2 allele burden (85% reduction; p < 0.0001).
        • Significantly reduced white blood cell (p < 0.01) and platelet counts (p < 0.05).
        • Significantly reduced liver weights (p < 0.05).
        • Reduced bone cellularity and led to normal myeloid differentiation in the bone marrow.
        • Significant decreases in MYC (a known target of PIM kinase inhibition) and E2F (a known target of CDK4/6 inhibition) gene expression.
        • Significantly prolonged overall survival of mice treated with triple therapy compared with either ruxolitinib treatment or vehicle (p < 0.0001).
        • Significant reduction in bone marrow reticulin fibrosis in triple therapy-treated mice (p < 0.01) but not doublet-treated mice.

        Interestingly, disease burden was increased when doses of either LEE011 or PIM447 or all three agents were reduced. There was no significant difference in hemoglobin, hematocrit or platelet counts between ruxolitinib and triple combination in wildtype C57BL/6 mice; however, white blood cell counts were significantly reduced with triple therapy (p < 0.01), indicating limited effects on normal erythropoiesis and megakaryopoiesis.

        Primary MPN patient samples

        The triple combination significantly reduced colony formation compared with ruxolitinib (p < 0.05 and p < 0.01 in different panels) in primary MPN patient samples. When investigated in normal human CD34+ cord blood cells, triple combination could significantly reduce colony formation compared with ruxolitinib but could not eliminate it.

        Conclusion

        Overall, these novel results suggest that the triple mechanism-based combination regimen of JAK1/2, pan-PIM, and CDK4/6 inhibition offers greater efficacy and potentially improved therapeutic responses compared with ruxolitinib monotherapy and ruxolitinib-based doublets for patients with MPN. Combined JAK/PIM/CDK inhibition significantly attenuated polycythemia vera and myelofibrosis disease phenotypes, although it did not fully eradicate the disease during the treatment duration. Notably, this approach could also better control MPN stem cell activity, contrary to JAK inhibition.

        1. Rampal RK, Pinzon-Ortiz M, Somasundara AVH, et al. Therapeutic efficacy of combined JAK1/2, pan-PIM, and CDK4/6 inhibition in myeloproliferative neoplasms. Clin Cancer Res. DOI: 10.1158/1078-0432.CCR-20-4898

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