Combining p38MAPK Inhibitors with a Second Targeted Agent Enhances Blockade of Inflammatory Signaling-Mediated Survival in Acute Myeloid Leukemia Cells
Authors: Kurtz Stephen E , Eide Christopher A. , Dubey Narain P. , Kaempf Andy , McWeeney Shannon K. , Tognon Cristina E. , Mori Motomi , Druker Brian J. , Tyner Jeffrey W. , Agarwal Anupriya 
Published: October 2019

Abstract
Background : Various pro-inflammatory and stress-related stimuli activate p38 mitogen-activated protein kinase (p38MAPK), triggering cascades of cell proliferation, differentiation, and apoptosis signaling. We have previously found that inflammatory cytokines promote growth and survival in primary cells from acute myeloid leukemia (AML) patients. The downstream mediator of inflammatory pathways is p38MAPK, and blocking this regulator with kinase inhibitors abrogates inflammation signaling in AML cells.
Methods : We used a functional ex vivo screening assay to identify small-molecule targeted inhibitors and inhibitor combinations demonstrating selective efficacy across broad categories of leukemia. Primary mononuclear cells isolated from leukemia patients (n=408) were plated in the presence of the p38MAPK inhibitor doramapimod or combinations of doramapimod with a second targeted agent that inhibits or effects a non-overlapping biological pathway. A 7-point concentration series was evaluated for both single agent inhibitors and combinations. Leukemia specimens from 408 unique patients were classified into 5 diagnosis subgroups including acute myeloid leukemia (AML; n=206), acute lymphoblastic leukemia (ALL; n=42), chronic lymphocytic leukemia (CLL; n=115), chronic myeloid leukemia (CML; n=16) and myeloproliferative neoplasms/myelodysplastic syndrome (MPN or MDS/MPN; n=29). IC50 and AUC values were derived from probit-based regression for each response curve. Mutational status for FLT3-ITD and NPM1 were compiled from clinical labs or by capillary electrophoresis using a QiaXcel instrument. Disease status was obtained from clinical chart review. For a subset of AML samples, RNAseq analysis was obtained in parallel to drug sensitivity testing. Single and combination drug treatment IC50 and AUC values were compared within groups by Friedman test, across groups by Kruskal-Wallis test, and with continuous variables by Spearman rank correlation.
Results : Among diagnostic subgroups tested, AML specimens showed the lowest median IC50 for doramapimod (1.71 uM), with 75 of 206 samples tested (36%) exhibiting IC50 values < 0.5 uM. In contrast, CLL specimens were significantly less sensitive (median IC50: 4.73 uM), with 27 of 115 samples tested (23%) showing IC50< 0.5 uM. For ALL, CML, and MDS/MPN subgroups, median IC50 values were 10, 3.19, and 6.78 uM, respectively; this translated to 17% of ALL, 19% of CML, and 38% of MDS/MPN samples with IC50< 0.5 uM. Doramapimod was also tested in combination with inhibitors of histone deacetylase (panobinostat), cyclin-dependent kinases 4/6 (CDK4/6; palbociclib), bromodomain and extra-terminal (BET) domain (JQ1), and BCL2 (venetoclax). Doramapimod combinations with panobinostat or JQ1 did not show enhanced efficacy compared to either single agent. However, combining doramapimod with palbociclib or venetoclax resulted in significantly enhanced efficacy compared to each single agent (median IC50: 0.014 and 0.075 uM, respectively; p<0.0001). Similar results were obtained using AUC as a drug effect measure. Of the 206 AML samples tested, 75% were sensitive (IC50 values < 0.5 uM) to both of these combinations, whereas 8% and 14% were sensitive to doramapimod in combination with only palbociclib or venetoclax, respectively. Sensitivity to combinations of doramapimod and palbociclib or venetoclax was not significantly associated with either age or gender. For the doramapimod + palbociclib combination, drug sensitivity (AUC) was correlated with gene expression for p38MAPKδ(Spearman r: -0.25). For doramapimod + venetoclax, sensitivity was correlated with expression of BCL2, MCL1, p38MAPKγ (Spearman r: -0.53, 0.23, and -0.33, respectively). Further analysis to align drug sensitivities with additional clinical and genetic features and inflammatory gene expression for AML patient samples is in progress.
Conclusions : AML patient specimens demonstrate ex vivo sensitivity to inhibition of p38MAPK, and this efficacy is enhanced when combined with inhibitors of either CDK4/6 or BCL2, suggesting dual inhibition of these pathways may extend clinical utility among patients with genetically heterogeneous leukemia.

Disclosures
Druker: Celgene: Consultancy; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Bristol-Meyers Squibb: Research Funding; Cepheid: Consultancy, Membership on an entity's Board of Directors or advisory committees; Beta Cat: Membership on an entity's Board of Directors or advisory committees; ALLCRON: Consultancy, Membership on an entity's Board of Directors or advisory committees; Aptose Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Aileron Therapeutics: Consultancy; Millipore: Patents & Royalties; McGraw Hill: Patents & Royalties; Henry Stewart Talks: Patents & Royalties; GRAIL: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals: Research Funding; Third Coast Therapeutics: Membership on an entity's Board of Directors or advisory committees; Oregon Health & Science University: Patents & Royalties; Fred Hutchinson Cancer Research Center: Research Funding; Monojul: Consultancy; ARIAD: Research Funding; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; Patient True Talk: Consultancy. Tyner:Genentech: Research Funding; Aptose: Research Funding; Janssen: Research Funding; Array: Research Funding; Incyte: Research Funding; Takeda: Research Funding; AstraZeneca: Research Funding; Constellation: Research Funding; Gilead: Research Funding; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees.