Aurora Kinase Inhibition Overcomes Primary Venetoclax Failure and Leads to Synthetic Lethality in BCL2-Positive Lymphomas Via Upregulation of P53/P21/BAX Axis
Authors: Mani Rajeswaran , Benrashid Samon , Teague Sarah E , Jaros Scott C , Yang Hsih-Te , Druhan Lawrence J , Foureau David M , Steuerwald Nury M , Ghosh Nilanjan , Copelan Edward A. , Durden Donald L. , Avalos Belinda , Park Steven I. 
Published: November 2021

Abstract

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 &lt; 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 &lt; 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 &gt;2 and FDR &lt; 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 &gt; 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.