BCL-2 Inhibition As a Synthetic Lethal Approach To Target Isocitrate Dehydrogenase Mutations In Acute Myeloid Leukemia Stem Cells

Mutations in isocitrate dehydrogenase (IDH) 1 and 2 occur in about 15% of acute myeloid leukemia (AML) patients. Studies of paired samples at diagnosis and relapse have demonstrated that IDH mutations, in contrast to FLT3 or RAS mutations, are stable during disease evolution. This finding indicates that a small population of cells harboring IDH mutations can persist in remission and eventually contribute to relapse. Therapeutic strategies that eradicate this IDH-mutated population have the potential to result in long-term remission.

Small molecule inhibitors specific for the mutant IDH enzymes have recently been developed (Wang et al Science 2013), but it is not known if they are effective in targeting primary AML cells including the leukemic stem cell (LSC) compartment in vivo. We sought an alternative approach to target IDH-mutated cells based on the concept of non-oncogene addiction which refers to the increased dependence on a subset of non-mutated genes for survival in response to activation of a specific oncogene. This dependency can be exploited therapeutically by inhibiting the activity of these non-oncogenes resulting in selective elimination of malignant cells, a phenomenon known as synthetic lethality (SL).

In an effort to identity SL targets against IDH mutations, we performed a pooled lentiviral RNA interference (RNAi) screen to search for genes that, when inhibited, led to the selective elimination of mutant IDH1 expressing cells. Our lentiviral short hairpin RNA (shRNA) library consisted of 27,500 unique shRNAs targeting 5,043 human genes of relevance to cancer biology. Each shRNA was tagged with a unique barcode sequence which permitted downstream identification by sequencing. The lentiviral shRNA library was transduced into a human AML cell line engineered to express mutant IDH1 (R132H) under the control of a doxycycline-inducible promoter. Following transduction, the cells were cultured in doxycycline to deplete shRNAs that were synthetic lethal to mutant IDH1. The relative abundance of each shRNA was subsequently determined by high-throughput sequencing of the barcode. Using a stringent algorithm designed to minimize false positive hits, the prosurvival gene BCL-2 was identified as one of the top SL hits. We confirmed that RNAi-mediated knockdown of BCL-2 expression was selectively lethal to mutant IDH expressing AML cell lines and further demonstrated that exposure to a cell-permeable form of (R)-2-hydroxyglutarate, the oncometabolite produced by mutant IDH, was sufficient to induce BCL-2 dependence. Similarly, pharmacologic inhibition of BCL-2 with ABT-199, a novel orally bioavailable and highly specific inhibitor of BCL-2 (Souers et al Nature Medicine 2013), was significantly more toxic to mutant IDH expressing AML cell lines than isogenic cell lines with wildtype (WT) IDH.

We next investigated the impact of IDH mutation status on ABT-199 sensitivity of primary AML cells and found that FACS-purified blasts with IDH mutations were 10-fold more sensitive to ABT-199 than blasts with WT IDH in ex vivo culture conditions. Normal cord blood hematopoietic stem and progenitor cells were highly resistant to ABT-199 treatment ex vivo suggestive of a wide therapeutic index. To demonstrate in vivo activity, we treated immunodeficient NOD/SCID/IL2Rγ-null (NSG) mice engrafted with primary human IDH-mutated leukemic cells with either ABT-199 at a dose of 100 mg/kg/day or vehicle control for 7 consecutive days by oral administration. Bone marrow engraftment analysis before and after treatment showed a 10 to 20-fold reduction in leukemic burden in ABT-199 treated mice, whereas no difference was seen in vehicle-treated mice. Importantly, bone marrow cells collected from ABT-199 treated mice failed to engraft in secondary transplant recipients indicative of a loss of LSC activity. In separate experiments, lentiviral transduction of BCL-2 shRNA vectors into IDH-mutated primary AML cells to knockdown BCL-2 expression impaired their engraftment in NSG mice, further validating the detrimental effect of BCL-2 inhibition on IDH-mutated LSCs.

In summary, our results indicate that IDH mutations increase BCL-2 dependence in leukemic cells including LSCs and identify a subgroup of patients that is likely to respond to pharmacologic BCL-2 inhibition. Our data provide the preclinical rationale for investigating the use ABT-199 in this patient subgroup in clinical trials.