Mutant Ikzf1, Kras ^G12D , and Notch1 cooperate in T lineage leukemogenesis and modulate responses to targeted agents

Abstract

Mice that accurately model the genetic diversity found in human cancer are valuable tools for interrogating disease mechanisms and investigating novel therapeutic strategies. We performed insertional mutagenesis with the MOL4070LTR retrovirus in Mx1-Cre, Kras G12D mice and generated a large cohort of T lineage acute lymphoblastic leukemias (T-ALLs). Molecular analysis infers that retroviral integration within Ikzf1 is an early event in leukemogenesis that precedes Kras G12D expression and later acquisition of somatic Notch1 mutations. Importantly, biochemical analysis uncovered unexpected heterogeneity, which suggests that Ras signaling networks are remodeled during multistep tumorigenesis. We tested tumor-derived cell lines to identify biomarkers of therapeutic response to targeted inhibitors. Whereas all T-ALLs tested were sensitive to a dual-specificity phosphoinosityl 3-kinase/mammalian target of rapamycin inhibitor, biochemical evidence of Notch1 activation correlated with sensitivity to γ-secretase inhibition. In addition, Kras G12D T-ALLs were more responsive to a MAP/ERK kinase inhibitor in vitro and in vivo. Together, these studies identify a genetic pathway involving Ikzf1, Kras G12D , and Notch1 in T lineage leukemogenesis, reveal unexpected diversity in Ras-regulated signaling networks, and define biomarkers of drug responses that may inform treatment strategies.

Deletion-based mechanisms of Notch1 activation in T-ALL: key roles for RAG recombinase and a conserved internal translational start site in Notch1

“…25,26 However, because 3 of the 6 lines with type 1 deletions we analyzed expressed apparently normal forms of Ikaros, loss of Ikaros function does not appear to be essential for activation of transcription from the 3Ј end of Notch1.…”

Preclinical efficacy of MEK inhibition in Nras-mutant AML

“…PD901 and GDC-0941 were administered at the maximum tolerated doses previously established in this strain background (F1 C57BL/6 3 129Sv/Jae). 24,[32][33][34] Leukemias #6730 and #8064 are classified as transplantable MPNs 35 because recipient mice develop progressive leukocytosis, retain residual erythropoiesis, and have a median survival of 70 days (supplemental Figure 3A-B). These MPNs represent a more aggressive disease than the indolent MPNs developed in Nras-mutant mice without retroviral insertions that are not transplantable to sublethally irradiated recipients.…”

Hematopoiesis and leukemogenesis in mice expressing oncogenic NrasG12D from the endogenous locus

“…Genomic DNA was extracted from the bone marrow and/or spleen of mice that developed AML, and the host-viral junction fragments were cloned and mapped as described. [20][21][22] DNA purification and southern blot analysis Genomic DNA was purified from hematologic tissues using PUREGENE DNA Isolation Kit (Gentra Systems) according to the manufacturer's protocol. A probe containing MOL4070LTR long-term repeat (LTR) sequences was purified from a sequence-verified vector.…”

“…15,16 By contrast, Mx1-Cre, Kras G12D pups that were injected with the same MOL4070LTR viral stock and treated with an identical poly I:C protocol did not spontaneously developed acute leukemia before dying from MPD at approximately 4 months of age. 21 Transplanting bone marrow from these animals induced AML in a small percentage of the recipients, with the majority developing T lineage acute lymphoblastic leukemia with prolonged latency. 21 Together, these observations raise the question of how to reconcile the profound effects of Kras G12D expression in the myeloid compartment with the potent ability of a "weaker" Nras G12D mutation to cooperate in AML induction.…”

“…21 Transplanting bone marrow from these animals induced AML in a small percentage of the recipients, with the majority developing T lineage acute lymphoblastic leukemia with prolonged latency. 21 Together, these observations raise the question of how to reconcile the profound effects of Kras G12D expression in the myeloid compartment with the potent ability of a "weaker" Nras G12D mutation to cooperate in AML induction. We speculate that this may be true because Kras G12D drives cellular proliferation and differentiation so strongly that it does not cooperate effectively with mutations that perturb self-renewal.…”