Momelotinib Is a Highly Potent Inhibitor of FLT3-Mutant AML

Approximately one-third of AML patients harbor kinase activating mutations in FLT3. Several small-molecule first generation FLT3 tyrosine kinase inhibitors (TKIs) have been evaluated in the last two decades, but none could induce a durable response possibly due to poor pharmacokinetics and target selectivity. Second generation FLT3 inhibitors such as, quizartinib, gilteritinib, and crenolanib, were designed for greater selectivity with a narrow kinome-profile and better pharmacokinetic properties, but they failed to induce a durable response. Possibly due to intrinsic resistance conferred by growth factor signaling in the bone marrow resident leukemic cells, which serve as a reservoir to develop resistance resulting to disease relapse. Activation of Jak2 signaling by chemokines and cytokines from the stroma have been reported to confer TKI refractoriness. Recent studies from Martin Carroll's group showed that both GMCSF and IL-3 confer resistance by activating JAK2 signaling, which can be suppressed by combined FLT3 and JAK2 inhibition. However, so far, it is not established whether upfront FLT3/Jak2 inhibition will provide durable response. For instance, AML patients who achieved complete remission lacking FLT3-ITD clones showed better overall survival than patients with measurable MRD, suggesting that eradicating the FLT3-ITD clones will have a deeper response with better overall survival. Thus, an ideal FLT3 inhibitor should be able to suppress FLT3-ITD resistant mutants, and growth factor activated JAK2 signaling while sparing c-KIT receptor and hERG to avoid myelosuppression and cardiotoxicity, respectively. Here we show that Jak2 inhibitor, momelotinib, is an equipotent type-I FLT3 inhibitor (Fig 1A-D). Biochemical and structural modeling revealed that it binds to an active conformation of FLT3 kinase. Therefore, like gilteritinib, it efficiently suppresses the resistance conferred by activation loop mutations (Fig1 E-H). Moreover, its lack of activity against c-KIT and inhibition of ACVR1 provides additional benefit in alleviating myelosuppression and anemia, which is commonly observed with currently used JAK2 and FLT3 inhibitors (ruxolitinib, fedratinib, and quizartinib). Perhaps more interestingly, momelotinib efficiently suppressed the disease in a preclinical model of AML using NSGS mice which recapitulates cytokine induce refractoriness as usually observed in clinical setting (Fig1 I-L). Our preclinical data provide evidence that momelotinib is an equipotent dual JAK2/FLT3 inhibitor and suppresses resistance conferred by both activation-loop mutations and growth-factor signaling. These data provide evidence that momelotinib treatment will have clinical activity in FLT3-mutated AML. Thus, warrants its clinical evaluation. Figure 1 Figure 1. Disclosures Starczynowski: kurome Inc: Consultancy.

Dual targeting of JAK2 and ERK interferes with the myeloproliferative neoplasm clone and enhances therapeutic efficacy

Myeloproliferative neoplasms (MPN) show dysregulated JAK2 signaling. JAK2 inhibitors provide clinical benefits, but compensatory activation of MAPK pathway signaling impedes efficacy. We hypothesized that dual targeting of JAK2 and ERK1/2 could enhance clone control and therapeutic efficacy. We employed genetic and pharmacologic targeting of ERK1/2 in Jak2V617F MPN mice, cells and patient clinical isolates. Competitive transplantations of Jak2V617F vs. wild-type bone marrow (BM) showed that ERK1/2 deficiency in hematopoiesis mitigated MPN features and reduced the Jak2V617F clone in blood and hematopoietic progenitor compartments. ERK1/2 ablation combined with JAK2 inhibition suppressed MAPK transcriptional programs, normalized cytoses and promoted clone control suggesting dual JAK2/ERK1/2 targeting as enhanced corrective approach. Combined pharmacologic JAK2/ERK1/2 inhibition with ruxolitinib and ERK inhibitors reduced proliferation of Jak2V617F cells and corrected erythrocytosis and splenomegaly of Jak2V617F MPN mice. Longer-term treatment was able to induce clone reductions. BM fibrosis was significantly decreased in MPLW515L-driven MPN to an extent not seen with JAK2 inhibitor monotherapy. Colony formation from JAK2V617F patients’ CD34+ blood and BM was dose-dependently inhibited by combined JAK2/ERK1/2 inhibition in PV, ET, and MF subsets. Overall, we observed that dual targeting of JAK2 and ERK1/2 was able to enhance therapeutic efficacy suggesting a novel treatment approach for MPN.

JAK inhibition for murine HLH requires complete blockade of IFNg signaling and is limited by toxicity of JAK2 inhibition

Hemophagocytic lymphohistiocytosis (HLH) is an inflammatory disorder in which numerous cytokines are elevated, though interferon gamma (IFN-g) is central to disease pathogenesis and a key therapeutic target. Experimental and early clinical reports have shown that ruxolitinib, a small molecule inhibitor of Janus kinases (JAKs) which are essential for cytokine signaling, may be therapeutic in HLH. In contrast, we found that intermittently administered ruxolitinib at various dose levels failed to prevent HLH development or treat established murine HLH. High doses of ruxolitinib blocked IFN-g signaling only transiently after administration, consistent with human pharmacokinetics, and only continuously administered drug could prevent HLH development or treat established HLH. Continuously administered ruxolitinib was therapeutic in only a narrow dose range and intermittently dosed ruxolitinib worsened survival and decreased bone marrow cellularity of animals concurrently treated with anti-IFN-g antibody, indicating a narrow therapeutic window and potential toxicity. As JAK2 is essential for hematopoietic cytokine signaling, we also tested a JAK1-selective inhibitor and observed therapeutic benefit without apparent toxicity, though it did not improve survival when combined with anti-IFN-g. We conclude that continuous blockade of IFN-g signaling is necessary for optimal control of HLH and that JAK2 inhibition may be toxic in this disorder.

2,4-SUBSTITUTED QUINAZOLINE AS JAK2 INHIBITOR: DOCKING AND MOLECULAR DYNAMICS STUDY

Objective: The involvement of Janus kinase2/signal transducer and activator of transcription (JAK2/STAT3) pathway reported in various solid tumors made authors study the conformational changes of JAK2-3e complex which was previously reported with a moderate percentage of In-vitro JAK2 inhibition. Methods: In this present study Compound 3e was reported with a moderate percentage of inhibition of JAK2 protein selected for performing molecular docking and molecular dynamics studies to elucidate the conformational changes with JAK2-3e complex. Docking studies were performed using ChemSketch to draw the structure of the compound and optimized/energy minimized using the Ligprep module of Schrodinger suite, employing optimized potentials for liquid simulations (OPLS-2005) force field. Molecular dynamics simulations were performed for 10 ns for complex using TIP4PEW water solvent model and neutralized by adding sodium ions. Results: Docking studies of Compound 3e which has been reported as one of the effective cytotoxic agents and a moderate percentage of In-vitro JAK2 inhibition among the series, showed H-bond interaction with leucine 855, serine936, aspartine994. Dock score and Ligand binding energy with protein suggested compound 3e has shown-4.049,-66.003 kcal/mol respectively. Molecular dynamics simulations elucidated the mechanistic insight of JAK-2 inhibition. The Root means square deviation (RMSD) pattern of both protein and ligands in the JAK2-3e complex observed to be different over 10 ns simulation. In the JAK2-3e complex, an exponential increase in RMSD of Cα and side-chain amino acids is observed during the first 1-3 ns simulation and is stabilized till 10 ns. During the 10 ns simulation, ligand 3e seems to be stable in the complex with an overall deviation<1 Å, despite a drastic increase between 1-3 ns. The ligand RMSD plot suggests that the ligand 3e remained intact within the binding site of the protein and longer time period simulation may elucidate the binding pattern and fate of ligand 3e. Conclusion: Results from molecular dynamics simulations elucidated the mechanistic insight of JAK-2 inhibition by 2, 4 disubstituted quinazoline compound that is N’(2-(4-nitrophenyl)quinazoline-4-yl) isonicotinohydrazide) and their binding phenomenon. Molecular docking studies further supported the elucidation of binding patterns of the molecules in the JAK-2 protein environment. Further simulations with a longer time period may provide deeper insights into ligand interactions in the protein environment. It is noteworthy to use compound 3e as a new scaffold for further development of multifunctional compounds.