Whole-genome sequencing analysis of newly diagnosed and relapsed multiple myeloma (MM) samples identified recurrent mutations in genes involved in the MAPK pathway, highlighting the potential of RAS/RAF/MEK/ERK signaling as a therapeutic target. Genomic studies identified translocations that involve IGH and set of partner genes MMSET, FGFR3, and CCND1 as primary events in MM. CDK4/CDK6 is overexpressed in MM, and CDK6 overexpression correlates with poor OS, suggesting that CDK4/6 are promising targets for MM therapy. Recent studies demonstrated synergistic activity of combined novel ERK1/2i inhibitor LY3214996 and CDK4/6i LY2835219 in solid tumors, but analogous studies have not been done in MM. Here we used preclinical models of MM to investigate inhibiting Erk1/2, CDK4/6, or both using ERK1/2i, CDK4/6i, or combination therapy. MM cell lines, RAS mutated or wild type (WT), were sensitive to ERK1/2i at IC50<0.5uM, and CDK4/6i at IC50<3uM. Synergistic effects of the Erk1/2i and CDK4/6i were noted in both RAS mutated and WT MM cell lines when ERK1/2i combined with CDK4/6i. Combination of ERK1/2i+CDK4/6i resulted in dose-dependent G0/G1 arrest in RAS mutated and WT MM cells. Similar effects were seen in RAS mutated cells treated with ERK1/2i or CDK4/6i as a single agent. ERK1/2i + CDK4/6i treatment triggered modest early apoptosis in RAS mutated MM cells, while in RAS WT MM cells this effect was more evident. Using dynamic BH3 profiling assay, we found that short-term treatment of MM cell with ERK1/2i and CDK4/6i led to increased overall mitochondrial priming in response to promiscuous BIM peptide in all MM cell lines. Even single agent treatment with ERK1/2i and CDK4/6i was able to enhance priming of RAS mutated or WT cells. Thus, ERK1/2i and CDK4/6i may activate mitochondrial apoptotic signaling in MM cells alone or in combination, consistent with observed synergistic cytotoxicity. HD PBMC and ARH77 cells were tested as controls. These cells were resistant to ERK1/2i and CDK4/6i at a broad range of concentrations, suggesting a favorable therapeutic index. The clinical potential of CDK4/6i+ERK1/2i was supported by an in vivo study demonstrating a significant (P=0.0004) decrease of the MM burden in CDK4/6i+ERK1/2i treated mice, without adverse effects. Proliferation and apoptosis studies of PCs from MM patient BM samples in the presence and absence of autologous BMSC/BMSCI-CM suggest potent and strong synergistic effects of ERK1/2i+CDK4/6i in MM and may allow successful use in clinic.
To address the underlying mechanism of the synergism between Erk1/2i and CDK4/6i, we evaluated their cellular and transcriptional activity in MM cells. Gene expression profiling showed significant downregulation of RAS and CDK4/6 signaling pathway genes in MM cells as a result of ERK1/2i and CDK4/6i treatment at specific concentration ratios (3:1/1:3). Further evaluation of functional effects of ERK1/2i and CDK4/6i, alone or in combination, demonstrated that the synergistic effect of these inhibitors in MM cells is achieved through inhibition of p-S6, downregulation of c-myc, and correlate with ERK1/2i+CDK4/6i induced cell arrest in the G1 cell cycle phase. We noted increased ERK1/2 phosphorylation, which generally results in compensatory activation of parallel signaling pathways or in the loss of negative feedback. Regardless, ERK1/2i+CDK4/6i retained the inhibitory activity of the downstream signaling network, as demonstrated by the inhibition of cytoplasmic (p-RSK1) and nuclear (c-myc) targets of ERK at protein and mRNA levels. Treatment with ERK1/2i+CDK4/6i significantly decreased the levels of p-Rb and E2F1, downstream targets of CDK4/6. Recent studies shown that, in addition to cell cycle regulation, CDK4 and CDK6 induce tumorigenesis through regulation of inflammatory cytokines that are induced via NFκB pathway activation. CDK4/6i functional effects on MM cells cannot be limited to cell cycle arrest, CDK4/6i might also inhibit cytokines, which are produced in MM cells by NFκB activation.
Overall, we shown that ERK1/2i+CDK4/6i induced cell proliferation and led to the key target molecule (p-c-myc, p-RSK, p-S6, p-RB, and E2F1) downregulations suggesting on-target activity of these inhibitors in MM cells. Importantly, our studies demonstrate strong synergistic anti-MM activity with ERK1/2+CDK4/6 therapy, providing a preclinical framework for clinical trials to improve patient outcome in MM.
Disclosures
Letai: Novartis: Research Funding; AbbVie: Consultancy; AstraZeneca: Consultancy; Zentalis: Membership on an entity's Board of Directors or advisory committees; Flash Therapeutics: Membership on an entity's Board of Directors or advisory committees; Dialectic: Membership on an entity's Board of Directors or advisory committees; Chugai: Other: Lecture Fees. Anderson:Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics..
Pre-Clinical Validation of a Novel Erk1/2 and CDK4/6 Inhibitor Combination in Multiple Myeloma (MM)
