Investigation of MLN4924 Alone and in Combination with Bcl-2 Inhibitors in Hematopoietic Stem Cells and Progenitors in Acute Myelogenous Leukemia

MLN4924, a novel chemotherapeutic agent designed to inhibit the Nedd8 Activating Enzyme (NAE), has shown promise in early clinical testing in relapsed and refractory Acute Myeloid Leukemia (AML). NAE inhibition prevents transfer of the Nedd8 protein to Cullin Ring Ligases (CRLs), leading to their inactivation and subsequent accumulation of the protein substrates they normally ubiquitinate and target for degradation by the proteasome. In malignant cells treated with MLN4924, various CRL substrates accumulate and induce a cytotoxic effect. Importantly, the substrates responsible for these effects vary among different malignancies depending on basal gene expression patterns.

In order to shed light on the mechanism of MLN4924 anti-leukemic effects and identify potential mechanisms of resistance, the effects of MLN4924 on AML cell lines and primary AML samples were examined in vitro and in vivo. These studies identified c-Myc as the critical substrate governing the cytotoxic effect. In particular, we observed c-Myc transactivation of the PMAIP1 gene, leading to expression of pro-apoptotic protein Noxa, which functions to neutralize Mcl-1, activate Bak and Bax to allow release of cytochrome c from the outer mitochondrial membrane, and induce apoptosis. Importantly, MLN4924-induced Noxa upregulation was prevented by c-Myc shRNA; and MLN4924-induced killing was markedly diminished by Noxa siRNA. In further experiments, a synergistic cytotoxic effect was seen when MLN4924 was applied to AML cell lines in combination with agents that inhibit anti-apoptotic proteins other than Mcl-1, such as BH3 mimetics ABT-263 (inhibits Bcl-2, Bcl-x_L, and Bcl-2) and ABT-199 (inhibits Bcl-2). Because expression of anti-apoptotic proteins within leukemia samples and between different AMLs is variable, and Mcl-1 upregulation often contributes to drug resistance and relapse in AML, this combination may be advantageous in several clinical settings.

To follow up on the effects we observed with MLN4924 alone and in combination with BH3 mimetics, we have designed a multi-parameter flow cytometry based assay to assess response to these agents among hematopoietic stem cells (HSCs) and progenitor populations in primary leukemia bone marrow isolates treated ex vivo. Pilot studies conducted in mononuclear cell fractions containing normal HSCs and hematopoietic progenitors isolated from umbilical cord blood did not undergo apoptosis in response to MLN4924. Ongoing studies aim to specifically assess whether MLN4924 alone or in combination with other anti-leukemic agents induce apoptosis or alter expression of CD47 (an anti-phagocytic protein frequently upregulated in AML) in HSCs and hematopoietic progenitor cell populations within primary AML bone marrow isolates.

As our understanding of how HSCs and hematopoietic progenitors contribute to AML initiation and progression advances, the need to target various populations will emerge and may vary depending on disease stage. It will be critical to ascertain whether HSCs and other populations are sensitive or resistant to agents such as MLN4924 and others in order to formulate and apply efficient targeted therapies.