T cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer that affects children and adults. Modern chemotherapy has improved clinical outcome but relapse and non-specific cytotoxicity are still problematic. Targeted therapy with specific inhibitors is highly desired but a better understanding of the aberrant biochemical pathways and pivotal molecules herein is required to reach this goal. ~50% of patient T-ALL patients show aberrantly active Ras signals. Until recently, the molecular players were unknown. We uncovered that T-ALL have two major mechanisms of abnormal Ras signaling: via overexpression of the Ras exchange factor Rasgrp1 or via oncogenic mutations in Ras (like K-RasG12D). Rasgrp1 overexpression occurs in ~55% of all pediatric T-ALL patients, mutations in KRAS in ~10%. We uncovered that Rasgrp1 continuously activates Ras that this is somehow counterbalanced by RasGAPs, and that cytokine receptor stimulation tips the balance in favor of active Ras. In 2013 we also reported (i) that myeloid leukemic cells with mutations in KRAS require Rasgrp3 and (ii) that Rasgrp molecules are autoinhibited and require 2nd messengers produced by Phospholipase C??(PLC?) for activation. In summary, these findings imply that Rasgrp's are critical components of leukemogenic Ras signals, are positioned downstream of cytokine receptor signaling, depend on PLC??for their activation, and are counterbalanced by RasGAPs. The mechanism of cytokine-Ras signaling, the molecular roles of RasGAPs, Rasgrp's, and PLC??herein, and the potential therapeutic effect of PLC??inhibiton in T-ALL are all unknowns. We obtained novel mechanistic insights through the development of innovative tools. We optimized a novel Ras activation assay to measure flux in the Ras GDP/GTP cycle that suggests critical buffering by RasGAPs. We optimized a quantitative, high-throughput method of phospho-flow combined with barcoding and a novel pINDUCER system for Dox-indicible shRNA. We established growth characteristics of Rasgrp1 and K-RasG12D T-ALL transplanted into nude mice. We can analyze primary patient T-ALL transplanted into recipient mice. Lastly, we developed an entirely novel genetic mouse model with overexpression of Rasgrp1 in bone marrow cells that leads to T-ALL and will compare this model to a genetic K-RasG12D model. Our biochemical-, cell biological-, and in vivo- approaches will reveal molecular insights into this novel but uncharacterized cytokine receptor-Rasgrp signaling pathway that is counterbalanced by RasGAP Ras inactivators (Aim 1) and will establish the molecular role of PLC??in Rasgrp-Ras-Ras effector activation (Aim 2).
In Aim 3, we will explore PLC??inhibition using pINDUCER or small molecule inhibitors in preclinical trials in the three above-mentioned mouse models. We anticipate that our studies will provide significant molecular insights into the basic science of leukemogenic signals but also provide translational insights in the therapeutic potential of PLC??inhibition that could impact clinical therapy forT-ALL in the future.
T cell leukemia is a disease with aberrantly active biochemical pathways that is currently treated with nonspecific chemotherapy because molecularly targeted therapy is not yet available. The goal of this project is to characterize the molecular mechanism of cytokine-induced Ras activation, to understand the role of a PLC?-Rasgrp module that appears critical herein as well as the role of RasGAPs as suppressors of this module, and to explore the effects of molecular interference with PLC??as a means of targeted therapy. Our comprehensive research plan utilizes established T cell leukemia lines, high-throughput quantitative biochemistry, novel mouse models for T cell leukemia, and patient T-ALL samples transplanted into NSG mice and will provide mechanistic insights into cytokine-induced PLC?-Rasgrp-Ras signaling and into the effects of molecular inhibition of this frequently dysregulated pathway, which will be of relevance to the mission of the NIH and of interest to a broad audience of cancer researchers.
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