Dual Targeting of MDM2 and BCL2/Bclxl Demonstrates Potent Synergistic Activity in High-Risk Adult Acute Lymphoblastic Leukemia

Despite the use of both intensive multi-modal cytotoxic therapeutic regimens and novel immune-based therapies in adult acute lymphoblastic leukemia (ALL) outcomes remain dismal, with five-year survival rates less than 45%. As such, there is an urgent need to identify novel therapies to improve patient outcomes. While TP53 mutations are infrequent in ALL, alternative mechanisms such as MDM2 overexpression or CDKN2A/B deletion may otherwise phenotypically disrupt and circumvent normal p53 function. We previously demonstrated encouraging pre-clinical activity of p53-MDM2 antagonist idasanutlin (RG7388) in diverse high-risk primary and patient-derived xenograft (PDX) ALL samples at concentrations well-below clinically attainable plasma levels [Bell et al., ASH 2020]. However, clinical studies reveal only modest responses to idasanutlin monotherapy in various hematological malignancies, emphasizing the need to identify effective drug combinations.

To systematically identify combination treatments which enhance cytotoxic idasanutlin activity, we performed comprehensive high-throughput phenotypic combination screening against 1971 FDA-approved and clinically advanced drugs. Extensive use of an ex vivo coculture system of ALL blasts and hTERT-immortalized mesenchymal stem cells (MSCs) was employed to support growth and survival of the ALL blasts, complemented by high-content image analysis to discriminate and enumerate the respective cell populations. Using the relapsed NALM6 B-ALL cell line and a panel of 6 PDX ALL samples - including relapsed and high-risk cytogenetic subtypes - we iteratively identified drugs (n=10) exhibiting significant synergistic and potent interaction with idasanutlin at concentrations well-below respective drug peak plasma concentrations reported in patients (Bliss synergy δ > 10). These included clinical ALL drugs dexamethasone and daunorubicin, proteasome inhibitor carfilzomib, BCL2/BCLxL inhibitor navitoclax, and histone deacetylase inhibitors romidepsin and pracinostat. Counter-screening of each drug candidate in a NALM6 TP53-isogenic cell model confirmed on-target p53-dependent combination activity (p<0.05). Further interrogation of each drug candidate was performed across a broad landscape of drug combination ratios with idasanutlin in primary (n=5) and PDX samples (n=6), including high-risk KMT2A-rearranged, ABL class T-ALL, and ETP-ALL. Combination of idasanutlin with navitoclax exhibited both the greatest and most consistent synergistic interaction (δ = 24.7±8.7, n=11) of the candidate combinations (n=10) across an extensive landscape of dose combinations; additionally alluding to synergy indiscriminate of ALL subtype.

Further characterizing the cytotoxic antileukemic activity of navitoclax, single-agent exposure achieved low nanomolar range, dose-dependent ex vivo sensitivity in a panel of B- (n=10) and T-ALL (n=4) primary and PDX samples, with half maximal inhibitory concentrations (IC₅₀) in the range of 3 to 11 nM (mean IC₅₀ = 6.4 nM) - almost 1000-fold lower than clinically attainable concentrations [Roberts et al., BJH2015]. While both idasanutlin and navitoclax effectively induced apoptosis in their own right, combination exposure markedly enhanced cell death. In an exceptionally high-risk TCF3-HLF PDX sample, 24 hours exposure to the drug combination increased annexin-V positive cells by 37.3±5.0% and 33.3±3.5% as compared to either drug (IC₅₀s, p<0.0001). In the NALM6 and RS4;11 relapsed ALL cell lines, combination exposure significantly increased annexin-V positive cells by at least two-fold (p<0.001) and enhanced apoptotic hallmark products cleaved poly(ADP-ribose) polymerase (PARP) (p<0.01) and cleaved caspase-3 as compared to either drug alone (IC₅₀s). Mechanistically, we found protein levels of MCL-1-binding pro-apoptotic sensitizer NOXA to be up to 3-fold increased in combination-treated blasts (p<0.05), potentially inhibiting compensatory anti-apoptotic MCL-1 activity - a known clinical resistance factor to BH3 mimetics.

Together, we provide strong evidence that concurrent targeting of MDM2-p53 binding and BCL2/BCLxL leads to potent and synergistic enhancement of apoptotic cell death in a range of high-risk ALL subtypes. The proposed combination of two clinical-stage compounds could have considerable positive clinical impact for the treatment of adult ALL.

Moorman:Amgen: Honoraria.