Investigation of Acyl Protein Thioesterase 1 and 2 as Potential Therapeutic Targets in NRAS Driven Leukemias

Noemi Zambetti and Amanda Long
Somatic NRAS mutations are highly prevalent in human cancers including acute myeloid leukemia (AML) and melanoma. The lack of effective therapies directed against this molecular lesion is a substantial barrier to improving clinical outcomes. Recent efforts in the Shannon laboratory that target effector molecules downstream of Ras, have shown promise, but inhibition of oncogenic RAS itself may constitute a more comprehensive and effective therapy for treating cancers driven by this prevalent oncogene.

Our work aims to investigate the possibility of inhibiting N-RAS oncogenic signaling activity by exploiting NRAS’s dependence on a dynamic cycle of depalmitoylation and repalmitoylation for proper function. This approach may allow selective targeting of cancer cells with oncogenic NRAS mutations, as signaling from WT K-RAS4b does not require palmitate turnover. We are examining the efficacy of selective inhibitors of enzymes responsible for RAS depalmitoylation in primary cells expressing oncogenic N- or KRAS. We are complementing these chemical inhibitors studies with shRNA-based genetic experiments to verify the relevant biological targets of small-molecule activity, as well as the general viability of these enzymes as therapeutic targets for further drug discovery.

Therapeutic Response and Resistance in Oncogenic Ras Driven AML

Ben Huang and Eugene Hwang
Oncogenic NRAS and KRAS mutations occur in 20% of acute myeloid leukemia (AML) implicating Ras as critical driver in AML. We performed retroviral insertional mutagenesis in NRas and KRas mutant mice to generate genetically diverse panels of transplantable, primary mouse AMLs driven by N-RasG12D or K-RasG12D. We treated cohorts of recipient mice transplanted with independent primary AMLs in vivo with kinase inhibitors that target downstream Ras effector pathways including MAPK and PI3K. In a majority of the AMLs, we observe a modest drug response to MAPK-pathway inhibition without clonal evolution or the development of a drug-resistant phenotype. A minority of the AMLs responded dramatically to treatment in vivo, but ultimately relapsed and acquired resistance to the drug. We are studying the mechanisms of resistance in these primary mouse leukemias and identifying pathways that may modulate sensitivity to these targeted agents. These studies provide a rationale for targeted combination therapy in Ras-driven AML and may elucidate novel mechanisms of resistance that are relevant to human disease.

Mechanisms of Response and Resistance in T-cell Acute Lymphoblastic Leukemia

Anica Wandler
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease characterized by aberrant proliferation of T-lymphoblasts. Recent data has demonstrated a high incidence of both Ras pathway mutations and NOTCH1 mutations in human T-ALLs. Previous work in our lab used retroviral insertional mutagenesis (RIM) to generate T-ALLs in both wild type mice, and those expressing oncogenic KrasG12Dfrom the endogenous locus. Treatment of these mice with targeted inhibitors of downstream Ras pathway components uncovered variability in both the initial response and acquired resistance to therapy. The goal of my project is to determine the mechanisms which determine how T-ALLs respond to drug treatment using several approaches. First, I will use whole exome sequencing to interrogate the “outlier” T-ALLs which show robust de novo sensitivity or resistance to therapy as a method of identifying genes which determine drug response. I am also investigating how mechanisms of response and resistance are affected by the type of therapy administered. To address this, I will perform a pre-clinical trial in the T-ALLs generated by RIM to examine response to glucocorticoids, a conventional chemotherapy drug, alone and in combination with a targeted therapy. Finally, I am examining potential effects of Notch1-independent mechanisms of T-ALL pathogenesis on response to therapy using cell lines derived from RIM-induced T-ALLs that do not exhibit Notch1 mutations.

Functional Analysis of Leukemia-Associated Chromosome 7 Deletions in the Mouse

Jasmine Wong and Ashley Ngo
Monosomy 7 (-7) and deletion 7q (del(7q)) are among the most common cytogenetic alterations found in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Cytogenetic analysis of patients who developed myeloid malignancies with del(7q) uncovered a commonly deleted segment (CDS) within chromosome band 7q22, suggesting that this region plays an important role in leukemogenesis. Genome sequencing of primary patient specimens showed that biallelic inactivation of individual 7q genes is remarkably infrequent in cases of AML or MDS with -7/del(7q). These data strongly support haploinsufficiency for multiple 7q genes as cooperating with mutations occurring elsewhere in the genome in the pathogenesis of MDS/AML with -7/del(7q). In order to understand the consequences of chromosome 7 deletions in an in vivo setting and to develop accurate model systems for biologic and preclinical studies, we used chromosome engineering to generate strains of mice with MB scale deletions in the DNA region syntenic to human chromosome 7q22. We are currently characterizing these mice to understand the consequences of the loss of this region on hematopoiesis and leukemogenesis. These mouse models are also useful for testing new therapies, and to study the biology of chromosome 7 deletions in cooperation with other genetic alterations frequently seen in MDS and AML.