Jon Akutagawa

Sustained MEK inhibition abrogates myeloproliferative disease in Nf1 mutant mice

Children with neurofibromatosis type 1 (NF1) are predisposed to juvenile myelomonocytic leukemia (JMML), an aggressive myeloproliferative neoplasm (MPN) that is refractory to conventional chemotherapy. Conditional inactivation of the Nf1 tumor suppressor in hematopoietic cells of mice causes a progressive MPN that accurately models JMML and chronic myelomonocytic leukemia (CMML). We characterized the effects of Nf1 loss on immature hematopoietic populations and investigated treatment with the MEK inhibitor PD0325901 (hereafter called 901). Somatic Nf1 inactivation resulted in a marked expansion of immature and lineage-committed myelo-erythroid progenitors and ineffective erythropoiesis. Treatment with 901 induced a durable drop in leukocyte counts, enhanced erythropoietic function, and markedly reduced spleen sizes in mice with MPN. MEK inhibition also restored a normal pattern of erythroid differentiation and greatly reduced extramedullary hematopoiesis. Remarkably, genetic analysis revealed the persistence of Nf1-deficient hematopoietic cells, indicating that MEK inhibition modulates the proliferation and differentiation of Nf1 mutant cells in vivo rather than eliminating them. These data provide a rationale for performing clinical trials of MEK inhibitors in patients with JMML and CMML.

A MEK Inhibitor Abrogates Myeloproliferative Disease in Kras Mutant Mice

Inhibiting the Raf/MEK/ERK pathway reverses the harmful effects of oncogenic Kras on hematopoietic differentiation, suggesting a strategy for treating myeloproliferative neoplasms. A MEKanistic Strategy for Beating Leukemia Overactivity of the Ras master signaling molecule has been implicated in both juvenile and chronic myelomonocytic leukemias (JMML and CMML). Despite its involvement in these leukemias, it has proven difficult to block either oncogenic Ras or its downstream signaling components. To address this issue, Lyubynska et al. used a mouse model with a mutation in the Kras gene (KrasG12D) that recapitulates features of the human myeloproliferative neoplasms JMML and CMML. They crossed two existing engineered strains to obtain the Mx1-Cre, KrasG12D mouse, which rapidly develops a progressive myeloproliferative neoplasm that is characterized by an increase in white blood cells (leukocytosis), an enlargement of the spleen (splenomegaly), and a lowered red blood cell count (anemia). Primary hematopoietic progenitor cells from the bone marrow of these mice display an overactive Raf/mitogen-activated or extracellular signal–regulated protein kinase kinase (MEK)/extracellular signal–regulated kinase (ERK) signaling pathway, which might be deregulated by oncogenic Ras; the authors therefore wondered whether blocking the downstream components of this pathway would be sufficient to block the effects of mutant Ras. So, they treated their 8-week-old Mx1-Cre, KrasG12D mice (with well-established myeloproliferative neoplasms) with PD0325901, a potent inhibitor of MEK, which is a signaling molecule that operates downstream of Ras. Compared to untreated mice, mice that received the MEK inhibitor demonstrated reduced leukocyte counts, disappearance of anemia, reduced spleen size, and prolonged survival—all of which indicate that PD0325901 reduces the severity of myeloproliferative disease. Lyubynska et al. attributed this positive effect to the ability of the MEK inhibitor to modulate the differentiation of bone marrow hematopoietic progenitor cells carrying the KrasG12D mutation, rather than boosting the proliferation of normal bone marrow progenitor cells. Although the effect of the Kras mutation might not be completely eliminated by MEK inhibition, the value of this new therapeutic strategy lies in reducing the symptoms caused by myeloproliferative neoplasms. Conventional chemotherapy exerts a purely antiproliferative effect on the rogue mutant hematopoietic progenitor cells, but PD0325901, in addition to restoring normal proliferation and differentiation programs for mutant myeloid progenitor cells, also helped to “rebalance” the hematopoietic system in vivo, despite continued KrasG12D expression. This intriguing study suggests that MEK inhibitors might be of clinical benefit for treating patients with JMML and CMML. 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 or extracellular signal–regulated protein kinase kinase (MEK), a downstream component of the Ras signaling network, to address how deregulated Raf/MEK/ERK (extracellular signal–regulated kinase) signaling drives neoplasia 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 neoplasms and further suggest that MEK inhibition could be a useful way to ameliorate functional hematologic abnormalities in patients with CMML and JMML.

Subclonal mutations in SETBP1 confer a poor prognosis in juvenile myelomonocytic leukemia

Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasm of childhood associated with a poor prognosis. Recently, massively parallel sequencing has identified recurrent mutations in the SKI domain of SETBP1 in a variety of myeloid disorders. These lesions were detected in nearly 10% of patients with JMML and have been characterized as secondary events. We hypothesized that rare subclones with SETBP1 mutations are present at diagnosis in a large portion of patients who relapse, but are below the limits of detection for conventional deep sequencing platforms. Using droplet digital polymerase chain reaction, we identified SETBP1 mutations in 17/56 (30%) of patients who were treated in the Childrens Oncology Group sponsored clinical trial, AAML0122. Five-year event-free survival in patients with SETBP1 mutations was 18% ± 9% compared with 51% ± 8% for those without mutations (P = .006).

Targeting the PI3K/Akt pathway in murine MDS/MPN driven by hyperactive Ras

Chronic and juvenile myelomonocytic leukemias (CMML and JMML) are myelodysplastic/myeloproliferative neoplasia (MDS/MPN) overlap syndromes that respond poorly to conventional treatments. Aberrant Ras activation because of NRAS, KRAS, PTPN11, CBL and NF1 mutations is common in CMML and JMML. However, no mechanism-based treatments currently exist for cancers with any of these mutations. An alternative therapeutic strategy involves targeting Ras-regulated effector pathways that are aberrantly activated in CMML and JMML, which include the Raf/MEK/ERK and phosphoinositide-3′-OH kinase (PI3K)/Akt cascades. Mx1-Cre, KrasD12 and Mx1-Cre, Nf1flox/− mice accurately model many aspects of CMML and JMML. Treating Mx1-Cre, KrasD12 mice with GDC-0941 (also referred to as pictilisib), an orally bioavailable inhibitor of class I PI3K isoforms, reduced leukocytosis, anemia and splenomegaly while extending survival. However, GDC-0941 treatment attenuated activation of both PI3K/Akt and Raf/MEK/ERK pathways in primary hematopoietic cells, suggesting it could be acting through suppression of Raf/MEK/ERK signals. To interrogate the importance of the PI3K/Akt pathway specifically, we treated mice with the allosteric Akt inhibitor MK-2206. This compound had no effect on Raf/MEK/ERK signaling, yet it also induced robust hematologic responses in Kras and Nf1 mice with MPN. These data support investigating PI3K/Akt pathway inhibitors as a therapeutic strategy in JMML and CMML patients.

PLC-γ and PI3K Link Cytokines to ERK Activation in Hematopoietic Cells with Normal and Oncogenic Kras

Targeting signaling pathways upstream of oncogenic Ras may have therapeutic benefit in the treatment of leukemia. Target Upstream of Oncogenic Ras Members of the K-Ras family of small guanosine triphosphatases mediate signaling by cytokine and growth factor receptors to activate extracellular signal–regulated kinase (ERK), leading to cellular proliferation. Mutant K-Ras molecules, for example, K-RasG12D, accumulate in the active form and are associated with certain leukemias. Through flow cytometric analysis of phosphorylated proteins in mouse bone marrow cells, Diaz-Flores et al. showed that ERK activation downstream of K-RasG12D required cytokine receptor–dependent activation of phospholipase C–γ (PLC-γ) and phosphoinositide 3-kinase (PI3K) signaling. Treatment of mice with a clinically available PI3K inhibitor reduced ERK activation in cells expressing K-RasG12D, suggesting that molecules upstream of oncogenic Ras may provide therapeutic targets against some cancers. Oncogenic K-Ras proteins, such as K-RasG12D, accumulate in the active, guanosine triphosphate (GTP)–bound conformation and stimulate signaling through effector kinases. The presence of the K-RasG12D oncoprotein at a similar abundance to that of endogenous wild-type K-Ras results in only minimal phosphorylation and activation of the canonical Raf–mitogen-activated or extracellular signal–regulated protein kinase kinase (MEK)–extracellular signal–regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)–Akt–mammalian target of rapamycin (mTOR) signaling cascades in primary hematopoietic cells, and these pathways remain dependent on growth factors for efficient activation. We showed that phospholipase C–γ (PLC-γ), PI3K, and their generated second messengers link activated cytokine receptors to Ras and ERK signaling in differentiated bone marrow cells and in a cell population enriched for leukemia stem cells. Cells expressing endogenous oncogenic K-RasG12D remained dependent on the second messenger diacylglycerol for the efficient activation of Ras-ERK signaling. These data raise the unexpected possibility of therapeutically targeting proteins that function upstream of oncogenic Ras in cancer.

Genome-wide DNA methylation is predictive of outcome in juvenile myelomonocytic leukemia

Juvenile myelomonocytic leukemia (JMML) is a myeloproliferative disorder of childhood caused by mutations in the Ras pathway. Outcomes in JMML vary markedly from spontaneous resolution to rapid relapse after hematopoietic stem cell transplantation. Here, we hypothesized that DNA methylation patterns would help predict disease outcome and therefore performed genome-wide DNA methylation profiling in a cohort of 39 patients. Unsupervised hierarchical clustering identifies three clusters of patients. Importantly, these clusters differ significantly in terms of 4-year event-free survival, with the lowest methylation cluster having the highest rates of survival. These findings were validated in an independent cohort of 40 patients. Notably, all but one of 14 patients experiencing spontaneous resolution cluster together and closer to 22 healthy controls than to other JMML cases. Thus, we show that DNA methylation patterns in JMML are predictive of outcome and can identify the patients most likely to experience spontaneous resolution.Juvenile myelomonocytic leukemia (JMML) is an aggressive disease with limited options for treatment. Here, the authors utilize DNA methylation based subgroups in JMML to predict clinical outcome.

Abstract 332: Targeting BCL-2 in hypodiploid acute lymphoblastic leukemia

Hypodiploid Acute Lymphoblastic Leukemia (ALL), is a high-risk subtype of ALL characterized by multiple chromosomal losses with an event-free survival To identify potential therapeutic targets we undertook a biochemical characterization of primary patient samples and cell lines, assessing the activation status of over 25 proteins involved in signaling pathways commonly deregulated in leukemia. Our biochemical characterization identified 1) constitutive activation of the PI3K pathway and the Bcl-2 survival pathway, and 2) accumulation of p53 that ultimately revealed that nearly 90% of low hypodiploid B-ALL patients harbor mutations in TP53 (Holmfeldt, et al., Nat. Genetics, 2013). We then set out to explore the therapeutic potential of targeting each of the deregulated pathways. We used multiple inhibitors and a library containing 94 compounds currently in early phase trials. Our studies showed high sensitivity to Bcl-2 inhibition, using ABT 263, ABT 737 and ABT 199, and partial sensitivity to PI3K inhibition. Surprisingly, these leukemia were relatively insensitive to inhibition of the Ras/MAPK signal transduction pathway, despite a high frequency of Ras pathway genomic alterations. Proliferation and apoptosis assays, performed in hypodiploid and non-hypodiploid leukemia cell lines (including both lymphoid and myeloid cell lines) showed a dramatic efficacy of targeting the BCL-2 pathway in hypodiploid leukemias. These results were replicated in primary patient samples xenografted into immunodeficient mice that were subjected to these drugs in vitro. A detailed biochemical characterization of the 12 members of the Bcl-2 family, together with a BH3 profiling to assess priming to apoptosis, has allowed us to determine the mechanisms underlying this sensitivity and efficacy on this leukemia. Our overall work has provided critical insights into the essential deregulated pathways underlying the pathogenesis of hypodiploid ALL. We are now continuing our investigations to identify novel strategies to treat these patients by using in vivo xenograft models of hypodiploid leukemia. We expect that our pre-clinical studies will provide further support for the therapeutic benefit of treating these leukemias with Bcl-2 inhibitors to improve survival for these young patients who suffer unacceptably high relapse rates from current therapies. Citation Format: Ernesto Diaz-Flores, Julie Weng, Jon Akutagawa, Linda Holmfeldt, Triona Chonghaile, Anthony Letai, Charles Mullighan, Benjamin Braun, Mignon Loh. Targeting BCL-2 in hypodiploid acute lymphoblastic leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 332. doi:10.1158/1538-7445.AM2014-332