T cell acute lymphoblastic leukemia/lymphoma (T-ALL) is an aggressive bone marrow cancer in children and adults, and chemotherapy often fails for relapsing patients. Molecularly targeted therapy is hindered by heterogeneity in T-ALL and mechanistic details of the affected pathways in T-ALL are needed. Deregulation of Ras signals is common in T-ALL. Ras is genetically mutated to a constitutively active form in about 15% of all haematopoietic malignancies, but there is a range of other ways to augment signaling through the Ras pathway. Several groups including our own uncovered that RasGRP1 overexpression leads to T-ALL in mouse models and in pediatric T-ALL patients, and we reported that this Ras guanine nucleotide exchange factor, RasGRP1, cooperates with cytokines to drive leukemogenesis. In our recent study by Ksionda et al. we analyzed the molecular details of cytokine receptor-RasGRP1-Ras signals in T-ALL and compared these to signals from mutated Ras alleles, which yielded several surprising results. Examples are the striking differences in flux through the RasGDP/RasGTP cycle in distinct T-ALL or unexpected differences in wiring of the Ras signaling pathway between T-ALL and normal developing T cells, which we will discuss here.

Oncogenic mutations in RAS genes, like KRAS^G12D or NRAS^G12D, trap Ras in the active state and cause myeloproliferative disorder and T cell leukemia (T-ALL) when induced in the bone marrow via Mx1CRE. The RAS exchange factor RASGRP1 is frequently overexpressed in T-ALL patients. In T-ALL cell lines overexpression of RASGRP1 increases flux through the RASGTP/RasGDP cycle. Here we expanded RASGRP1 expression surveys in pediatric T-ALL and generated a RoLoRiG mouse model crossed to Mx1CRE to determine the consequences of induced RASGRP1 overexpression in primary hematopoietic cells. RASGRP1-overexpressing, GFP-positive cells outcompeted wild type cells and dominated the peripheral blood compartment over time. RASGRP1 overexpression bestows gain-of-function colony formation properties to bone marrow progenitors in medium containing limited growth factors. RASGRP1 overexpression enhances baseline mTOR-S6 signaling in the bone marrow, but not in vitro cytokine-induced signals. In agreement with these mechanistic findings, hRASGRP1-ires-EGFP enhances fitness of stem- and progenitor- cells, but only in the context of native hematopoiesis. RASGRP1 overexpression is distinct from KRAS^G12D or NRAS^G12D, does not cause acute leukemia on its own, and leukemia virus insertion frequencies predict that RASGRP1 overexpression can effectively cooperate with lesions in many other genes to cause acute T-ALL.

Deregulated signal transduction is a cancer hallmark, and its complexity and interconnectivity imply that combination therapy should be considered, but large data volumes that cover the complexity are required in user-friendly ways. Here, we present a searchable database resource of synthetic lethality with a PI3 kinase signal transduction inhibitor by performing a saturation screen with an ultra-complex shRNA library containing 30 independent shRNAs per gene target. We focus on Ras-PI3 kinase signaling with T cell leukemia as a screening platform for multiple clinical and experimental reasons. Our resource predicts multiple combination-based therapies with high fidelity, ten of which we confirmed with small molecule inhibitors. Included are biochemical assays, as well as the IPI145 (duvelisib) inhibitor. We uncover the mechanism of synergy between the PI3 kinase inhibitor GDC0941 (pictilisib) and the tubulin inhibitor vincristine and demonstrate broad synergy in 28 cell lines of 5 cancer types and efficacy in preclinical leukemia mouse trials.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic cancer. Poly-chemotherapy with cytotoxic and genotoxic drugs causes substantial toxicity and more specific therapies targeting the underlying molecular lesions are highly desired. Perturbed Ras signaling is prevalent in T-ALL and occurs via oncogenic RAS mutations or through overexpression of the Ras activator RasGRP1 in ~65% of T-ALL patients. Effective small molecule inhibitors for either target do not currently exist. Genetic and biochemical evidence link phosphoinositide 3-kinase (PI3K) signals to T-ALL, PI3Ks are activated by Ras-dependent and Ras-independent mechanisms, and potent PI3K inhibitors exist. Here we performed comprehensive analyses of PI3K-Akt signaling in T-ALL with a focus on class I PI3K. We developed a multiplex, multiparameter flow cytometry platform with pan- and isoform-specific PI3K inhibitors. We find that pan-PI3K and PI3K γ-specific inhibitors effectively block basal and cytokine-induced PI3K-Akt signals. Despite such inhibition, GDC0941 (pan-PI3K) or AS-605240 (PI3Kγ-specific) as single agents did not efficiently induce death in T-ALL cell lines. Combination of GDC0941 with AS-605240, maximally targeting all p110 isoforms, exhibited potent synergistic activity for clonal T-ALL lines in vitro, which motivated us to perform preclinical trials in mice. In contrast to clonal T-ALL lines, we used a T-ALL cancer model that recapitulates the multi-step pathogenesis and inter- and intra-tumoral genetic heterogeneity, a hallmark of advanced human cancers. We found that the combination of GDC0941 with AS-605240 fails in such trials. Our results reveal that PI3K inhibitors are a promising avenue for molecular therapy in T-ALL, but predict the requirement for methods that can resolve biochemical signals in heterogeneous cell populations so that combination therapy can be designed in a rational manner.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic cancer. Poly-chemotherapy with cytotoxic and genotoxic drugs causes substantial toxicity and more specific therapies targeting the underlying molecular lesions are highly desired. Perturbed Ras signaling is prevalent in T-ALL and occurs via oncogenic RAS mutations or through overexpression of the Ras activator RasGRP1 in ~65% of T-ALL patients. Effective small molecule inhibitors for either target do not currently exist. Genetic and biochemical evidence link phosphoinositide 3-kinase (PI3K) signals to T-ALL, PI3Ks are activated by Ras-dependent and Ras-independent mechanisms, and potent PI3K inhibitors exist. Here we performed comprehensive analyses of PI3K-Akt signaling in T-ALL with a focus on class I PI3K. We developed a multiplex, multiparameter flow cytometry platform with pan- and isoform-specific PI3K inhibitors. We find that pan-PI3K and PI3K γ-specific inhibitors effectively block basal and cytokine-induced PI3K-Akt signals. Despite such inhibition, GDC0941 (pan-PI3K) or AS-605240 (PI3Kγ-specific) as single agents did not efficiently induce death in T-ALL cell lines. Combination of GDC0941 with AS-605240, maximally targeting all p110 isoforms, exhibited potent synergistic activity for clonal T-ALL lines in vitro, which motivated us to perform preclinical trials in mice. In contrast to clonal T-ALL lines, we used a T-ALL cancer model that recapitulates the multi-step pathogenesis and inter- and intra-tumoral genetic heterogeneity, a hallmark of advanced human cancers. We found that the combination of GDC0941 with AS-605240 fails in such trials. Our results reveal that PI3K inhibitors are a promising avenue for molecular therapy in T-ALL, but predict the requirement for methods that can resolve biochemical signals in heterogeneous cell populations so that combination therapy can be designed in a rational manner.