Targeting the DNA Damage Response Pathway in MLL- rearranged B-Acute Lymphoblastic Leukemias

Infant B-acute lymphoblastic leukemias (B-ALL) that harbor MLL-AF4 rearrangements are associated with a poor prognosis (event-free survival (EFS) of 35%-50%), especially when compared to non-MLL-rearranged ( MLL - r ) childhood ALL (EFS > 85%). Thus, effective therapies that ensure long-term durable responses are of extreme interest. Recently, the targeting of the DNA damage response pathway (DDR) has been shown to be cytotoxic to MLL-r acute myeloid leukemia (AML) cells with oncogene driven replicative stress. In order to determine if targeting of the DDR pathway would be efficacious in MLL-r B-ALLs, we tested a panel of human AML and ALL cell lines and found that all B-ALL cell lines (those with and without MLL-r ) were highly sensitive to ATR kinase inhibition. At 72 hours, IC₅₀s for MLL- r B-ALL cell lines (RS4;11, SEMK2, Hb11;19) were 2-3 fold lower than MLL-AF9 AML cell lines (Molm13, THP-1, Nomo1). Inhibition of ATR kinase induced double stranded DNA damage (as assessed by the increase in phospho-γH2AX levels), apoptosis and loss of the G2-M cell cycle checkpoint in a dose dependent manner in all cells treated, with B-ALL cells exhibiting these effects more rapidly and at lower doses.

In order to better understand the mechanism behind the increased sensitivity of MLL -r B-ALLs to ATR inhibition, we generated a highly aggressive, serially transplantable B-ALL by the retroviral overexpression of activating N-Ras^G12D mutant in bone marrow of an inducible knock-in MLL-Af4 murine model. Recipient mice injected with Mll-Af4/N-Ras^G12D pre-leukemic bone marrow cells developed an acute B-ALL (B220+CD43+IgM-) with short latency to development of disease (median 35 days). Furthermore, the resultant primary B-ALL was serially transplantable, with accelerated latency to secondary and tertiary disease. Multiple sequencing studies have indicated that while MLL -r B-ALL have relatively few additional somatic mutations, activating mutants of RAS seem to co-occur at a frequency close to 50%. Moreover, among MLL -r infant leukemias, those harboring mutant RAS mutations have even worse overall prognosis, further emphasizing the utility of our model.

We were able to detect increased basal levels of DNA damage and replicative stress in our Mll-Af4/Nras^G12D pre-leukemic cells and sensitivity to a small molecule inhibitor of ATR, AZ20. In vitro and in vivo treatment with AZ20 resulted in increased leukemia cytotoxicity. However, tertiary B-ALL transplanted mice eventually succumbed to disease following prolonged AZ20 treatment in vivo . As our model includes an activating mutation in N-Ras , we hypothesized that cells would be sensitive to the combination with small molecule inhibitors of downstream effectors of Ras. The combination of AZ20 and MEK inhibitor GSK212 (Trametinib) increased cytotoxicity and cell cycle arrest in vitro at concentrations well below the IC₅₀, as compared to single agent treatment. In vivo combination treatment of not only our mouse model but also of patient derived xenografts, harboring both mutations, exhibited decreased leukemia burden and significant prolonged survival compared to either AZ20 or Trametinib alone. These data suggest a potent combination therapy for the treatment of this subgroup of poor prognosis MLL-AF4 patients with RAS mutations.

To try to gain some further mechanistic insight, we conducted gene set enrichment analysis of RNA-sequencing of primary Mll-Af4/N-Ras^G12D leukemias and normal pro-B cells. Our analyses revealed significant negative correlations with gene signatures involving the G2/M checkpoint and ATR activation in response to replication stress, suggesting derangements in DDR pathways. We validated these observations with quantitative RT-PCR of ATR pathway members in murine mouse models of MLL-r AML and B-ALL. Our studies suggest that the observed sensitivity to ATR inhibition correlates to the overall expression level of ATR and ATR pathway members. We observed similar patterns of sensitivity and expression in the human cell lines we tested, as well as in an analysis of published gene expression data of infant and pediatric B-ALL patients. Altogether, our data indicates the inhibition of ATR in cells with oncogene driven replicative stress and abrogated ATR and ATR pathway expression, as part of an effective combination therapeutic strategy in this subtype of acute leukemia.