Mantle cell lymphoma (MCL) is an aggressive, rare, and difficult to treat subtype of non-Hodgkin's Lymphoma (NHL) that accounts for about 6% of all cases. Although there is no defined standard of care for MCL treatment, some combination of chemo-immunotherapy and rituximab maintenance with or without autologous stem cell transplantation is generally employed depending on the age and fitness of the patient. Despite recent development of novel therapeutics, there is inevitable disease relapse with progressively declining efficacy and increasing frequency of resistance with single agent targeted therapy. Here, we describe the novel multitarget inhibitor SRX3177 which simultaneously hits three oncogenic targets: phosphatidylinositol-3 kinase (PI3K), cyclin-dependent kinases 4 and 6 (CDK4/6), and the epigenetic reader protein BRD4. This in silico designed, thieno-pyranone (TP) scaffold-based small molecule inhibitor orthogonally disrupt three targets within the cancer cell with one agent. Targeting the cell cycle with small molecule inhibitors represents a reasonable attempt to treat MCL, as cell cycle-associated genes like ATM, TP53, CDKN2A, CCND1 and CDK4/6 are most frequently mutated in patients. Palbociclib is a well-known single agent CDK4/6 inhibitor that has been employed in both solid and hematological malignancies. Due to its cytostatic nature, treatment with single agent palbociclib often results in the emergence of treatment-resistant clones. Therefore, a combination strategy would theoretically be more effective and can overcome the development of resistance. Moreover, prolonged G1 arrest by CDK4/6 inhibition sensitizes lymphoma cells to PI3K inhibition, suggesting a synthetic lethality relationship between these two agents. Inhibiting the chromatin reader protein BRD4 causes downregulation of target genes c-MYC and BCL2, further increasing cytotoxic capabilities. Hence, we developed SRX3177 as a potent CDK4/6/PI3K/BRD4 triple inhibitor to synergistically inhibit cell cycle progression and induce cancer cell apoptosis. SRX3177 is an ATP competitive CDK4/6 inhibitor (IC 50: CDK4 = 2.54 nM, CDK6 = 3.26 nM), PI3K inhibitor (IC 50: PI3Kα = 79.3 nM, PI3Kδ = 83.4 nM), and BRD4 inhibitor (IC 50: BD1 = 32.9 nM, BD2 = 88.8 nM). We have tested the efficacy of SRX3177 against a panel of MCL cell lines and report that SRX3177 induces a strong antiproliferative activity with maximal IC 50 0f 340 nM in JeKo-1, 29 nM in Mino cells, and 630 nM for Rec-1 cells while IC 50 values for cell lines Granta and JVM-2 were 1.3 µM and 1.5 µM, respectively. Further, we show that SRX3177 is more potent to tumor cells than the individual PI3K (BKM120), BTK (Ibrutinib), BRD4 (JQ1), and CDK4/6 (palbociclib) inhibitors, and dual PI3K/BRD4 inhibitor SF2523 (backbone for SRX3177) in JeKo-1 cells. Next, we examine the cytotoxic effect of SRX3177 in ibrutinib/palbociclib resistant primary MCL cells. Our results show that SRX3177 triggers cytotoxic response at 500 nM and 1000 nM as compared to the lack of cytotoxicity of combination Ibrutinib and palbociclib at 150 nM and 1000 nM (Fig 1). SRX3177 induces a strong apoptotic response and cell cycle arrest in JeKo-1 and Mino cells at 24hrs. Annexin V/7AAD apoptosis staining confirmed the induction of PCD by SRX3177with increase in c-PARP. Western blot analysis shows SRX3177 treatment blocks both PI3K/AKT signaling and Rb phosphorylation. Moreover, analysis by chromatin immunoprecipitation revealed that SRX3177 effectively blocked BRD4 binding to both the promoter and enhancer of c-MYC (p≤0.01 and p≤0.001) and BCL2 (p≤0.05). SRX3177 also suppresses the c-MYC and BCL2 transcriptional program in both a time- and dose-dependent manner. Our findings also demonstrate a SRX3177-dependent reduction in c-MYC half-life via induction of proteasomal-mediated degradation. This degradation is associated with decreased phosphorylation of c-MYC at Ser62 and increased phosphorylation of c-MYC at Thr58 - indicative of differential regulation of c-MYC stability. Finally, we show that SRX3177 overcomes chronic ibrutinib resistance in Jeko-1 cells with a maximal IC 50 of 150 nM as compared to 64 µM with ibrutinib. Hence, the triple inhibitor SRX3177 has superior potency to ibrutinib in MCL cell lines and succeeds in overcoming ibrutinib-resistance at nanomolar doses. Taken together, our data supports the development of SRX3177 as a novel therapeutic agent for treatment of MCL. Figure 1 Figure 1. Disclosures Martin: ADCT: Consultancy. Park: Takeda: Research Funding; G1 Therapeutics: Consultancy; Teva: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Research Funding, Speakers Bureau; Gilead: Speakers Bureau; Rafael Pharma: Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board; Morphosys: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding. Durden: SignalRx Pharmaceuticals: Current holder of individual stocks in a privately-held company.
Venetoclax (VEN), a BCL2 specific inhibitor, has shown excellent clinical activities in various types of non-Hodgkin lymphoma, and it is FDA-approved in patients with chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL). However, its efficacy has been mostly disappointing in BCL2-positive (BCL2+) lymphomas harboring classic BCL2 rearrangements, including follicular lymphoma (FL), double-hit lymphoma (DHL), and triple-hit lymphoma (THL). The mechanism by which BCL2+ lymphomas evade BCL2 inhibition remains elusive although it has shown to be in part due to overexpression of anti-apoptotic proteins like BCLxL or MCL1. Aurora kinases (AURK) are serine threonine kinases involved in mitotic regulation and have functional role in stabilization of regulatory proteins such as MYC. Here in we investigated potential mechanisms of primary resistance to VEN and the effect of AURK inhibition in overcoming primary VEN failure in BCL2+ lymphomas. BCL2+ lymphoma cells, WSU-NHL (single hit; BCL2 only), DoHH2 (DHL; BCL2 and MYC), and VAL (THL; BCL2, MYC, and BCL6) were evaluated for cell viability (ATP quantification) and apoptosis (Annexin V/7AAD staining), after treatment with various concentrations of VEN with or without MLN8237 (AURK-A inhibitor), LY3295668 (AURK-A inhibitor), or AZD2811 (AURK-B inhibitor). Addition of an AURK-A or B inhibitor to VEN induced robust killing and displayed synergism only in BCL2+ but not in BCL2-negative Raji and Ramos Burkitt lymphoma cells (MYC only). AURK-A inhibition using MLN8237 was chosen for further in-depth functional analysis. Immunoblotting revealed increased caspase-3 cleavage in DoHH2 cells treated with VEN+MLN8237 combination than either agent alone. No significant changes in BCL2, BCLxL or MCL1 protein levels were noticed in DoHH2 and VAL cells after single or combined treatments. However, MLN8237 resulted in elevated levels of proapoptotic proteins BAX and PUMA. MYC degradation occurred later in cells after treatment with MLN8237 or combination implying that MYC degradation may be a delayed and independent effect. Furthermore, VEN+MLN8237 combination completely cleared tumors in two different BCL2+ lymphoma mouse models where mice were randomized into four groups and treated with vehicle, VEN, MLN8237, or VEN+MLN8237 combination via oral gavage for 15 days. First, in a DoHH2 DHL xenograft SCID mouse model, VEN+MLN8237 combination resulted in complete tumor regression and 100% tumor-free survival on day 100 (p < 0.0001; N=8/group) with no discernable toxicity, while all mice in other groups were euthanized due to disease progression within 45 days. Next, in a disseminated THL model using VAL cells intravenously infused into NCG mice, all animals receiving combination therapy survived with no evidence of disease on day 100 (p < 0.0001; N=6-8/group), while all except one in other groups were euthanized due to removal criteria, including hindlimb paralysis and weight loss, by day 60. To investigative the tumor response to BCL2 and AURK inhibitions, we performed transcriptome sequencing (RNA seq) of DoHH2 tumors harvested from SCID mice (N=6-7/group) treated for 3 days under the 4 conditions as described above. Comparison of VEN with VEN+MLN8237 combination identified 41 genes of which 33 increased and 8 decreased in combination therapy compared to VEN alone (Fold change >2 and FDR < 0.05). Most notably, CDKN1A (p21) level was decreased by 2-fold in VEN monotherapy compared to vehicle control while the concurrent inhibition of AURK-A by MLN8237 reversed this process by upregulating p21 by > 4-fold compared to VEN monotherapy. Ingenuity pathway analysis subsequently revealed that VEN+MLN8237 combination induced significant upregulation of p53/p21/BAX network. Additional assays confirmed an early characteristic downregulation of p53 protein levels in response to VEN treatment in BCL2+ lymphoma cells. The induction of p53, p21, PUMA, and BAX in VEN+MLN8237 combination was further confirmed by immunoblotting. In contrast, VEN reduced p21, PUMA and BAX expression levels compared to vehicle treated cells. p53 knockdown in DoHH2 cells resulted in similar resistance to VEN and combination treatment. Taken together these data suggest AURK inhibition overcomes downregulation of p53/p21/BAX axis by BCL2+ lymphomas in response to BCL2 inhibition, hence lay the groundwork for further evaluation of this combination in clinical settings. Figure 1 Figure 1. Disclosures Foureau: Cytognos: Honoraria; TeneoBio, Celgene: Research Funding. Ghosh: Pharmacyclics LLC, an AbbVie Company: Consultancy, Honoraria, Research Funding, Speakers Bureau; Seattle Genetics: Consultancy, Honoraria, Speakers Bureau; Epizyme: Honoraria, Speakers Bureau; Incyte: Consultancy, Honoraria; TG Therapeutics: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Speakers Bureau; Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; Genmab: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Speakers Bureau; ADC Therapeutics: Consultancy, Honoraria; Adaptive Biotech: Consultancy, Honoraria; AbbVie: Honoraria, Speakers Bureau; Karyopharma: Consultancy, Honoraria; Genentech: Research Funding. Copelan: Amgen: Consultancy. Durden: SignalRx Pharmaceuticals: Current holder of individual stocks in a privately-held company. Avalos: Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees; BMJ Best Practice: Patents & Royalties: Royalties from a co-authored article on evaluation of neutropenia. Park: Takeda: Research Funding; Rafael Pharma: Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board; Morphosys: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Research Funding, Speakers Bureau; Gilead: Speakers Bureau; G1 Therapeutics: Consultancy; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding; Teva: Consultancy, Membership on an entity's Board of Directors or advisory committees.
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