The NF1 tumor suppressor gene encodes a guanosine triphosphotase (GTPase)-activating protein that negatively regulates Ras signaling and is inactivated in a subset of juvenile myelomonocytic leukemias (JMMLs). Adoptive transfer of fetal liver cells from Nf1 mutant mice models JMML; however, this system has important limitations as a platform for performing biologic and preclinical studies. We have exploited the interferon-inducible Mx1-Cre transgene to ablate a conditional mutant Nf1 allele in hematopoietic cells. Somatic inactivation of Nf1 induces a myeloproliferative disorder with 100% penetrance that is associated with a subacute clinical course, tissue infiltration by myeloid cells, hypersensitivity to granulocyte-macrophage colony stimulating factor, hyperproliferation, and resistance to apoptosis. These Mx1-Cre, Nf1 flox/flox mice establish a tractable experimental model for testing therapeutics and for identifying mutations that cooperate with hyperactive Ras in myeloid leukemogenesis. IntroductionJuvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative disease (MPD) characterized by monocytosis, thrombocytopenia, splenomegaly, and malignant infiltration of the skin, lymph nodes, lungs, liver, and other organs (reviewed in Emanuel et al 1 and Arico et al 2 ). The clinical course is relentless, and bone marrow transplantation is the only treatment that cures more than 10% of patients. Selective hypersensitivity of granulocyte-macrophage colony-forming unit (CFU-GM) progenitors to granulocyte-macrophage colony-stimulating factor (GM-CSF) is an in vitro hallmark of JMML. 3,4 The incidence of JMML is increased more than 200-fold in children with neurofibromatosis type 1 (NF1) 5,6 ; this observation provided a starting point for elucidating the molecular basis of aberrant myeloid growth in this disorder. The NF1 gene encodes neurofibromin, a guanosine triphosphotase (GTPase)-activating protein (GAP) that negatively regulates p21 ras (Ras) output by accelerating GTP hydrolysis (reviewed in Boguski and McCormick,7 Bernards, 8 and Donovan et al 9 ). Analysis of JMML cells from children with NF1 revealed homozygous NF1 inactivation because of somatic loss of the normal allele, which is associated with hyperactive Ras. [10][11][12][13] Two groups used homologous recombination in embryonic stem cells to disrupt Nf1, the murine homolog of NF1. 14,15 Approximately 10% of heterozygous (Nf1 ϩ/Ϫ ) mutant mice spontaneously develop a MPD that resembles JMML during the second year of life. 14 Homozygous mutant (Nf1 Ϫ/Ϫ ) embryos fail around embryonic day 13 (E13) with cardiovascular defects 14,15 ; however, CFU-GM colonies derived from mutant fetal livers show hypersensitive growth in response to GM-CSF that is similar to human JMML cells. 11,16 Importantly, adoptive transfer of Nf1 Ϫ/Ϫ fetal liver cells consistently induces a JMML-like MPD in irradiated recipient mice. 16 Nf1 inactivation leads to deregulated growth in multiple hematopoietic compartments and confers a durable proliferative advantage i...

The Raf/MEK/ERK cascade is a therapeutic target in human cancers with deregulated Ras signaling, which includes tumours that have inactivated the Nf1 tumour suppressor1. Nf1 encodes neurofibromin, a GTPase activating protein that terminates Ras signalling by stimulating hydrolysis of Ras•GTP. We compared the effects of inhibitors of MEK in a myeloproliferative disorder (MPD) initiated by inactivating Nf1 in mouse bone marrow and in acute myeloid leukaemias (AMLs) in which cooperating mutations were induced by retroviral insertional mutagenesis. Here we show that MEK inhibitors are ineffective in MPD, but induce objective regression of many Nf1-deficient AMLs. Drug resistance developed due to outgrowth of AML clones that were present before treatment. We cloned clone-specific retroviral integrations to identify candidate resistance genes including Rasgrp1, Rasgrp4, and Mapk14, which encodes p38α. Functional analysis implicated increased RasGRP1 levels and reduced p38 kinase activity in resistance to MEK inhibitors. This approach represents a robust strategy for identifying genes and pathways that modulate how primary cancer cells respond to targeted therapeutics and for probing mechanisms of de novo and acquired resistance.

Chronic and juvenile myelomonocytic leukemias (CMML and JMML) are aggressive myeloproliferative neoplasms that are incurable with conventional chemotherapy. Mutations that deregulate Ras signaling play a central pathogenic role in both disorders, and Mx1-Cre, KrasLSL-G12D mice that express the Kras oncogene develop a fatal disease that closely mimics these two leukemias in humans. Activated Ras controls multiple downstream effectors, but the specific pathways that mediate the leukemogenic effects of hyperactive Ras are unknown. We used PD0325901, a highly selective pharmacological inhibitor of mitogen-activated protein kinase kinase (MEK), a downstream component of the Ras signaling network, to address how deregulated Raf/MEK/ERK signaling drives neoplasm formation in Mx1-Cre, KrasLSL-G12D mice. PD0325901 treatment induced a rapid and sustained reduction in leukocyte counts, enhanced erythropoiesis, prolonged mouse survival, and corrected the aberrant proliferation and differentiation of bone marrow progenitor cells. These responses were due to direct effects of PD0325901 on Kras mutant cells rather than to stimulation of normal hematopoietic cell proliferation. Consistent with the in vivo response, inhibition of MEK reversed the cytokine hypersensitivity characteristic of KrasG12D hematopoietic progenitor cells in vitro. Our data demonstrate that deregulated Raf/MEK/ERK signaling is integral to the growth of Kras-mediated myeloproliferative neoplasias, and further suggest that MEK inhibition could be a useful way to ameliorate functional hematologic abnormalities in patients with CMML and JMML.

Identifying the molecular basis for inherited cancer predispositions reveals genes that when mutated, play a critical role in the earliest stages of tumorigenesis. Although rare, inherited predispositions to myeloid leukemias have led to a greater understanding of pathways important for myeloid proliferation and maturation. In particular, elucidating why children with neurofibromatosis type 1 (NF1) and Noonan syndrome (NS) are predisposed to juvenile myelomonocytic leukemia (JMML) has uncovered a critical role of hyperactive Ras signaling in normal myeloid growth and leukemogenesis. Here, we review studies of human samples and experiments performed in genetically engineered strains of mice investigating the molecular and biochemical basis of aberrant growth in JMML. These strains model human disease features and provide an opportunity to investigate novel therapeutic strategies that may ultimately cure JMML and other myeloid malignancies characterized by hyperactive Ras.

Purpose This first-in-human phase I trial assessed the safety, tolerability, and preliminary anti-tumor activity of apitolisib (GDC-0980), a dual inhibitor of class I phosphatidylinositol-3-(PI3K) and mammalian target of rapamycin (mTOR) kinases. Experimental Design Once-daily (QD) oral apitolisib was administered to patients with solid tumors for days 1-21 or 1-28 of 28-day cycles. Pharmacokinetic and pharmacodynamic parameters were assessed. Results Overall, 120 patients were treated at doses between 2-70 mg. The commonest ≥G3 toxicities related to apitolisib at the recommended phase 2 dose (RP2D) at 40mg QD included hyperglycemia (18%), rash (14%), liver dysfunction (12%), diarrhea (10%), pneumonitis (8%), mucosal inflammation (6%), and fatigue (4%). Dose-limiting toxicities (one patient each) were G4 fasting hyperglycemia at 40 mg (21/28-schedule), and G3 maculopapular rash and G3 fasting hyperglycemia at 70 mg (21/28-schedule). The pharmacokinetic profile was dose-proportional. Phosphorylated serine-473 AKT levels were suppressed by ≥90% in platelet-rich plasma within 4 hours at the maximum tolerated dose (50 mg). Pharmacodynamic decreases in FDG-PET uptake of >25% occurred in 66% (21/32) of patients dosed at 40 mg QD. Evidence of single agent activity included ten RECIST partial responses (confirmed for peritoneal mesothelioma, PIK3CA mutant head- and-neck cancer, and three pleural mesotheliomas). Conclusion Apitolisib exhibited dose-proportional pharmacokinetics with target modulation at doses ≥16 mg. The RP2D was 40 mg QD 28/28-schedule; severe on-target toxicities were apparent at ≥40 mg, particularly pneumonitis. Apitolisib was reasonably tolerated at 30 mg, the selected dose for pleural mesothelioma patients given limited respiratory reserve. Modest but durable anti-tumor activity was demonstrated.

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NCT01564251.

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