Ari J. Firestone

Preclinical efficacy of MEK inhibition in Nras-mutant AML

Oncogenic NRAS mutations are highly prevalent in acute myeloid leukemia (AML). Genetic analysis supports the hypothesis that NRAS mutations cooperate with antecedent molecular lesions in leukemogenesis, but have limited independent prognostic significance. Using short hairpin RNA-mediated knockdown in human cell lines and primary mouse leukemias, we show that AML cells with NRAS/Nras mutations are dependent on continued oncogene expression in vitro and in vivo. Using the Mx1-Cre transgene to inactivate a conditional mutant Nras allele, we analyzed hematopoiesis and hematopoietic stem and progenitor cells (HSPCs) under normal and stressed conditions and found that HSPCs lacking Nras expression are functionally equivalent to normal HSPCs in the adult mouse. Treating recipient mice transplanted with primary Nras(G12D) AMLs with 2 potent allosteric mitogen-activated protein kinase kinase (MEK) inhibitors (PD0325901 or trametinib/GlaxoSmithKline 1120212) significantly prolonged survival and reduced proliferation but did not induce apoptosis, promote differentiation, or drive clonal evolution. The phosphatidylinositol 3-kinase inhibitor GDC-0941 was ineffective as a single agent and did not augment the activity of PD0325901. All mice ultimately succumbed to progressive leukemia. Together, these data validate oncogenic N-Ras signaling as a therapeutic target in AML and support testing combination regimens that include MEK inhibitors.

Dominant Role of Oncogene Dosage and Absence of Tumor Suppressor Activity in Nras-Driven Hematopoietic Transformation

UNLABELLED Biochemical properties of Ras oncoproteins and their transforming ability strongly support a dominant mechanism of action in tumorigenesis. However, genetic studies unexpectedly suggested that wild-type (WT) Ras exerts tumor suppressor activity. Expressing oncogenic Nras(G12D) in the hematopoietic compartment of mice induces an aggressive myeloproliferative neoplasm that is exacerbated in homozygous mutant animals. Here, we show that increased Nras(G12D) gene dosage, but not inactivation of WT Nras, underlies the aggressive in vivo behavior of Nras(G12D/G12D) hematopoietic cells. Modulating Nras(G12D) dosage had discrete effects on myeloid progenitor growth, signal transduction, and sensitivity to MAP-ERK kinase (MEK) inhibition. Furthermore, enforced WT N-Ras expression neither suppressed the growth of Nras-mutant cells nor inhibited myeloid transformation by exogenous Nras(G12D). Importantly, NRAS expression increased in human cancer cell lines with NRAS mutations. These data have therapeutic implications and support reconsidering the proposed tumor suppressor activity of WT Ras in other cancers. SIGNIFICANCE Understanding the mechanisms of Ras -induced transformation and adaptive cellular responses is fundamental. The observation that oncogenic Nras lacks tumor suppressor activity, whereas increased dosage strongly modulates cell growth and alters sensitivity to MEK inhibition, suggests new therapeutic opportunities in cancer.

KRAS Engages AGO2 to Enhance Cellular Transformation.

Oncogenic mutations in RAS provide a compelling yet intractable therapeutic target. Using co-immunoprecipitation mass spectrometry, we uncovered an interaction between RAS and Argonaute 2 (AGO2). Endogenously, RAS and AGO2 co-sediment and co-localize in the endoplasmic reticulum. The AGO2 N-terminal domain directly binds the Switch II region of KRAS, agnostic of nucleotide (GDP/GTP) binding. Functionally, AGO2 knockdown attenuates cell proliferation in mutant KRAS-dependent cells and AGO2 overexpression enhances KRAS(G12V)-mediated transformation. Using AGO2-/- cells, we demonstrate that the RAS-AGO2 interaction is required for maximal mutant KRAS expression and cellular transformation. Mechanistically, oncogenic KRAS attenuates AGO2-mediated gene silencing. Overall, the functional interaction with AGO2 extends KRAS function beyond its canonical role in signaling.

Monoallelic Loss of the Imprinted Gene Grb10 Promotes Tumor Formation in Irradiated Nf1 +/- Mice

Imprinted genes are expressed from only one parental allele and heterozygous loss involving the expressed allele is sufficient to produce complete loss of protein expression. Genetic alterations are common in tumorigenesis but the role of imprinted genes in this process is not well understood. In earlier work we mutagenized mice heterozygous for the Neurofibromatosis I tumor suppressor gene (NF1) to model radiotherapy-associated second malignant neoplasms that arise in irradiated NF1 patients. Expression analysis of tumor cell lines established from our mouse models identified Grb10 expression as widely absent. Grb10 is an imprinted gene and polymorphism analysis of cell lines and primary tumors demonstrates that the expressed allele is commonly lost in diverse Nf1 mutant tumors arising in our mouse models. We performed functional studies to test whether Grb10 restoration or loss alter fundamental features of the tumor growth. Restoring Grb10 in Nf1 mutant tumors decreases proliferation, decreases soft agar colony formation and downregulates Ras signaling. Conversely, Grb10 silencing in untransformed mouse embryo fibroblasts significantly increased cell proliferation and increased Ras-GTP levels. Expression of a constitutively activated MEK rescued tumor cells from Grb10-mediated reduction in colony formation. These studies reveal that Grb10 loss can occur during in vivo tumorigenesis, with a functional consequence in untransformed primary cells. In tumors, Grb10 loss independently promotes Ras pathway hyperactivation, which promotes hyperproliferation, an early feature of tumor development. In the context of a robust Nf1 mutant mouse model of cancer this work identifies a novel role for an imprinted gene in tumorigenesis.

KRAS insertion mutations are oncogenic and exhibit distinct functional properties

Oncogenic KRAS mutations introduce discrete amino acid substitutions that reduce intrinsic Ras GTPase activity and confer resistance to GTPase-activating proteins (GAPs). Here we discover a partial duplication of the switch 2 domain of K-Ras encoding a tandem repeat of amino acids G60_A66dup in a child with an atypical myeloproliferative neoplasm. K-Ras proteins containing this tandem duplication or a similar five amino acid E62_A66dup mutation identified in lung and colon cancers transform the growth of primary myeloid progenitors and of Ba/F3 cells. Recombinant K-RasG60_A66dup and K-RasE62_A66dup proteins display reduced intrinsic GTP hydrolysis rates, accumulate in the GTP-bound conformation and are resistant to GAP-mediated GTP hydrolysis. Remarkably, K-Ras proteins with switch 2 insertions are impaired for PI3 kinase binding and Akt activation, and are hypersensitive to MEK inhibition. These studies illuminate a new class of oncogenic KRAS mutations and reveal unexpected plasticity in oncogenic Ras proteins that has diagnostic and therapeutic implications.

Correction: Monoallelic Loss of the Imprinted Gene Grb10 Promotes Tumor Formation in Irradiated Nf1+/- Mice.

The corresponding author for this article is not listed. The corresponding author is Jean L. Nakamura (ude.fscu@arumakaN.naeJ). The editor’s affiliation is missing. Bruce Korf’s affiliation is: University of Alabama, Birmingham School of Medicine, United States of America.

Abstract B43: Requirement for PI3 kinase interaction in K-RasG12D-driven leukemogenesis.

Background: Somatic RAS mutations occur in approximately 30% of human cancers. Oncogenic Ras proteins activate multiple downstream effectors including Raf, PI3 kinase (PI3K), and Ral-GDS. The tyrosine 64 residue, which is located in the Switch II effector binding domain of Ras, is an important contact for binding PI3K catalytic subunits. To investigate the role of PI3K binding in hematopoeitic transformation in vivo, we created a “second site” Lox-STOP-Lox (LSL- KrasG12D/Y64G) knock in allele. Methods: LSL-KrasG12D and LSL-KRasG12D/Y64G mice on a 129/Sv background were crossed with C57BL/6 Mx1-Cre mice. These mice were injected with polyIpolyC (pIpC) at 21 days of age to excise the LSL casette, which was confirmed by QPCR. Colony growth potential of primary bone marrow cells was assessed by sacrificing six to ten week old Mx1-Cre;LSL-KrasG12D, Mx1-Cre;LSL-KrasG12D/Y64G, and wild type mice, isolating bone marrow, and plating 50,000 cells in methylcellulose media containing 0, 0.01, 0.1, 1, or 10 ng/ml GM-CSF. Activation of Ras effector pathways was assessed in macrophages derived from primary bone marrow cells harvested from 6-10 week old mice and grown in the presence of M-CSF. Results: As previously described, inducing oncogenic KrasG12D expression in the hematopoietic compartment of Mx1-Cre;KrasG12D mice results in death by three months of age from an aggressive myeloproliferative neoplasm (MPN) that accurately models chronic and juvenile myelomonocytic leukemias. MPN development is greatly attenuated in Mx1-Cre;LSL-KrasG12D/Y64G mice, which develop anemia beginning at six months of age, but do not display elevated blood leukocyte counts and have >90% survival at eight months. Bone marrow cells from Mx1-Cre;LSL-KrasG12D mice form myeloid progenitor colonies in methylcellulose without added cytokines and show profound hypersensitivity to GM-CSF. By contrast, G12D/Y64G doubly mutant progenitors require GM-CSF for colony formation, and display modestly elevated cytokine sensitivity compared to wild type progenitor cells. Whereas cultured Mx1-Cre;LSL-KrasG12D and Mx1-Cre;LSL-KrasG12D/Y64G macrophages contain markedly elevated levels of active Ras-GTP, they exhibit different patterns of effector pathway activation. In particular, basal levels of phosphorylated ERK and Akt (pERK and pAkt) are unexpectedly higher in Mx1-Cre;LSL-KRasG12D/Y64G macrophages, implicating the PI3K pathway in feedback responses to oncogenic Ras output. Conclusions: Our initial data support an important role of efficient PI3K activation in KrasG12D-driven leukemia and uncover unexpected complexity in cellular responses to Ras-GTP. We are continuing to monitor blood counts in aging Mx1-Cre;LSL-KrasG12D/Y64G mice and are analyzing effects of this mutation on eythroid differentiaion and on hematopoieitc stem and progentior cell (HSPC) populations. In addition, this novel conditional mutant allele will be a valuable genetic tool for assessing the importance of oncogenic-Ras-mediated PI3K signaling in other cancers with frequent somatic KRAS mutations including lung, pancreatic, and colon. Citation Format: Yasmine N. White, Ari J. Firestone, Kevin M. Shannon. Requirement for PI3 kinase interaction in K-RasG12D-driven leukemogenesis. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr B43.

Abstract LB-058: Novel interactions of the RAS oncoprotein

Approximately 30% of all cancers harbor activating mutations in the RAS family of small GTPase proteins, making it one of the most common oncogenic aberrations in humans. Normal RAS proteins (H, K or N-RAS) localize to the inner cell membrane and transduce extracellular growth signals by cycling between an “active” GTP-bound state and “inactive” GDP-bound state, through interactions with various “GTPase activating proteins” (GAPs) that promote RAS mediated GTP hydrolysis. Oncogenic mutants of RAS lose their catalytic activity or association with the GAP proteins, resulting in constitutively active GTP-bound state that signals through direct interactions with effector kinases like RAF and PI3K and activate the MEK/ERK and/or Akt, leading to activation of hallmark cancer pathways including growth factor independence, uncontrolled cell proliferation, evasion of apoptosis and immune responses, increased metabolism as well as metastases. Although RAS is the most frequently mutated gene driving multifarious pathways of oncogenesis, our knowledge of protein interactions involving RAS proteins have been largely limited to RAS binding domains in RAF/PI3K/RalGDS. Targeting mutant RAS proteins or its direct effectors, or pathways activated by RAS effectors remains a challenging endeavor for treating RAS driven cancers. Towards the goal of a thorough understanding of RAS biology through a comprehensive identification of its interactors, we performed IP-Mass Spectrometric analysis of pan-RAS immunoprecipitates from multiple cell lines. Interestingly in our experiments, apart from the well-known interactor RAF, we found evidence of several novel RAS interacting proteins, including many with DNA and RNA binding motifs. Our study validates these findings through cell-free protein interaction analyses and explores the possible biological effects of these novel RAS interactions in mutant KRAS driven cellular transformation. Note: This abstract was not presented at the meeting. Citation Format: Sunita Shankar, Rohit Malik, Vishal Kothari, Yasuyuki Hosono, Sethuramasundaram Pitchiaya, Shanker Kalyana-Sundaram, Anastasia Yocum, June Escara-Wilke, Harika Gundlapalli, Krishnapriya Chinnaswamy, Matthew Shuler, Anton Poliakov, Xiaoju Wang, Vishalakshi Krishnan, Yasmine White, Ari Firestone, Xuhong Cao, Saravana M. Dhanasekaran, Jeanne Stuckey, Gideon Bollag, Kevin Shannon, Nils G. Walter, Chandan Kumar-Sinha, Arul Chinnaiyan. Novel interactions of the RAS oncoprotein. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-058. doi:10.1158/1538-7445.AM2015-LB-058