Project Summary/Abstract
Given the persistently poor prognosis of acute myeloid leukemia (AML), diagnostic and therapeutic strategies
need to be developed to achieve significantly improved cure rates. While initial response rates and event-free-
survival have increased, most patients relapse and succumb to the disease. The regimen consisting of BCL-2
inhibitor venetoclax (VEN) in combination with a hypomethylating agent (HMA) (VEN/HMA) has revolutionized
AML therapy with complete remission rates, now ranging from 45 to 90%, accompanied by prolonged survival.
Notably, the underlying mechanism of action of VEN/HMA therapy resides in the inhibition of oxidative
phosphorylation (OXPHOS), especially in AML leukemia stem cells (LSCs). However, the majority of patients
receiving VEN/HMA eventually relapse, especially patients with high-risk characteristics including complex
cytogenetics and aberrant RAS and FLT3 signaling. The role of p53 in AML cell death is poorly understood.
We recently we reported that the inhibition of nuclear exporter XPO1 (CRM1) causes accumulation of p53 in
the nuclei of AML cells, and that dual inhibition of the ubiquitin E3 ligase MDM2 and XPO1 substantially
amplifies this activity leading to synergistic p53-mediated killing of AML cells, even of VEN/HMA resistant cells,
in vitro and in vivo. After dual MDM2 and XPO1 inhibition, a small fraction of surviving AML cells expressed
high levels of p21, a p53 target, LC3B, and the key integrated stress response (ISR) factor activated
transcription factor 4 (ATF4), which should render the residual AML cells vulnerable to BCL-2 inhibition. Indeed,
the triple combination of a MDM2, XPO1, and BCL-2 inhibitor resulted in the highest ATF4 protein levels and
the deepest cytoreduction. Interestingly, we found highly increased protein levels of OXPHOS complexes in
AML cells with acquired resistance to dual MDM2 and XPO1 inhibition in vivo suggesting OXPHOS activation.
This finding provided the rationale for overcoming this resistance mechanism with VEN/HMA in combination.
We hypothesized that 1) chronic p53 reactivation confers AML OXPHOS dependency thereby restoring
sensitivity to VEN-based therapy; and 2) the concomitant combinatorial treatment of MDM2, XPO1 inhibitors
with VEN/HMA efficiently suppresses AML and AML LSCs. The hypothesis will be examined with the
following Specific Aims (SAs). In SA 1, we will investigate the functional dependency on OXPHOS in AML
cells resistant to dual MDM2 and XPO1 inhibition. We will characterize the cellular responses and cell fates, at
the single-cell level, using high-parametric flow cytometry and mass cytometry (CyTOF) for LSCs and blasts
upon maximal p53 activation by dual inhibition of MDM2 and XPO1 with or without VEN/HMA, to assess
apoptosis and other modes of regulated cell death at multiple time points. In SA 2, we will examine the anti-
leukemia effects of dual MDM2 and XPO1 inhibition combined with VEN/HMA in AML and AML LSCs with
wild-type p53. The successful completion of the proposed research should provide a novel treatment approach
for VEN/HMA-resistant AML with non-genotoxic, targeted therapeutics.
