Kevin M. Shannon - US grants

DESCRIPTION: (adapted from the investigator's abstract) Children with the common inherited disorder neurofibromatosis, type 1 (NF1) are predisposed to myeloid leukemia, particularly juvenile chronic myelogenous leukemia (JCML). The NF1 gene (NF1) encodes a protein called neurofibromin that stimulates the GTPase activity of the p21 ras (Ras) family of signaling proteins. This activity of neurofibromin suggested that NF1 might function as a tumor-suppressor gene in myeloid cells by negatively regulating Ras. Genetic and biochemical data from the laboratory support this hypothesis. The investigators have developed a murine model to investigate the role of NF1 in myeloid growth control. This translational research project is based on laboratory data which suggest that deregulated signaling through the Ras pathway in response to granulocyte macrophage colony stimulating factor (GM-CSF) plays a central role in the over-proliferation of myeloid cells that is characteristic of murine NF1-/- fetal liver cells and JCML bone marrow cells. This proposal has 3 specific aims. The experiments proposed under aim 1 will provide a rigorous genetic test of the hypothesis that the deregulated growth of NF1-/- cells is GM-CSF-specific. The experiments proposed in aim 2 are based on genetic and biochemical evidence that hyperactive Ras plays a central role in the aberrant phenotype of murine Nf1-/- and human JCML cells. These mechanistic data suggest that drugs that target the Ras pathway are rational potential theraeputics for JCML and other myeloid leukemias. Mice reconstituted with Nf1-/- fetal liver cells provide a genetically-defined in vivo model system to test this hypothesis. The third experimental aim will involve characterizing the germ line and somatic NF1 mutations that exist in children with leukemia as these data may provide novel insights into mechanisms of normal growth control and tumorigenesis.

blood /lymphatic neoplasm; neoplasm /cancer genetics; gene mutation; chromosome deletion; tumor suppressor genes; acute myelogenous leukemia; neoplasm /cancer radiation therapy; genetic markers; neoplasm /cancer chemotherapy; molecular oncology; clinical research; artificial chromosomes; polymerase chain reaction; genetic mapping; human subject; human genetic material tag;

This translational research proposal addresses the epidemiology, biology and treatment of children with the related preleukemic conditions juvenile chronic myelogenous leukemia and bone marrow monosomy 7 syndrome (JCML/Mo7). These disorders share a number of major clinical features that suggest they are related pathogenically; however, the precise relationship between them is uncertain. Treatment results in children with JCML/Mo7 have been dismal even with allogeneic bone marrow transplantation. This proposal is based upon recent epidemiological findings linking specific obstetric and parental occupational risk factors with the development of myeloid leukemia during infancy; upon the observation that some children with JCML have show clinical responses to the differentiating agent cis retinoic acid; and upon molecular genetic and biochemical studies that implicate deregulated signaling through the p21 ras family of proteins in the pathogenesis of these malignant myeloid disorders. We will test the hypothesis that JCML/Mo7 are associated with genetic and acquired risk factors which correlate with specific molecular and cytogenetic alterations in patient bone marrows, and that these molecular markers will be of prognostic value and can be used to measure responses to treatment.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] This application is made to renew a component of the Mouse Models of Human Cancer Consortium (MMHCC) that focuses on modeling myeloid malignancies in the mouse. These diseases represent a substantial burden, and current treatment strategies are unsatisfactory. During the initial period of support, the applicant team has largely achieved our research goals, which involve engineering and characterizing new and existing murine models of myeloid malignancies, exploiting retroviral insertional mutagenesis to perform forward genetic screens to identify genes that contribute to leukemia initiation and progression, developing new technologies and classification systems for analyzing mouse myeloid malignancies, and generating resources to benefit the entire mouse cancer modeling research community. We have also made substantial contributions to the MMHCC though participation in many of its activities. This renewal will build upon and extend this work through integrated experiments to: (1) utilize retroviral insertional mutagenesis to discover and interrogate novel genes that contribute to leukemogenesis in the context of known leukemia-associated genetic lesions; (2) harness mice with segmental deletions that model the losses of 5q31 and 7q22 found in human myeloid leukemia for gene discovery and functional studies; and, (3) harness cytogenetic analysis, SKY, and expression profiling to address specific questions relevant to the pathogenesis and treatment of murine and human myeloid malignancies. We are also fully committed to continuing to develop the MMHCC as a resource for the cancer research community. The applicant team is comprised on the same group of 6 investigators that are currently contributing to these studies and have developed a track record of highly productive research interactions. [unreadable] [unreadable] [unreadable]

This application requests funds to purchase a cesium 137 irradiation source to operate within the barrier Animal Care Facility (ACF) at the Parnassus Heights campus of the University of California, San Francisco (UCSF). Parnassus Heights is the major site of teaching, research, and patient care at UCSF. Locating this instrument within the ACF will allow 11 co-investigators working in the areas of immunology, infectious disease, hematopoiesis, and cancer biology whose laboratories are located on the Parnassus campus to perform experiments which are integral to NIH-funded research projects and which involve transferring hematopoietic cells into irradiated mice. Acquisition of a cesium irradiator for the ACF will ensure that these studies are carried out efficiently, safely, and in a manner that follows well-established procedures for conducting working within the controlled environment of a barrier animal care facility. The applicant group has a excellent record of research productivity and a strong history of collaborating with each other. At present, there is no radiation source available for irradiating small animals at the Parnassus campus. Existing procedures are expensive, cumbersome, and inefficient as they involve housing mice at a different site from the investigator s laboratory or transferring animals to another site within San Francisco for radiation, and then transporting them back to the Parnassus campus. The instrument will be housed within the ACF, will add an important new component to the infrastructure of that facility, and will greatly enhance research efforts of UCSF investigators in addition to the applicant group. The application documents strong institutional support for the proposal.

Funding is requested to support the next meeting of the National Neurofibromatosis Foundation International Consortium for the Molecular and Cell Biology of NF1 and NF2 to be held June 4-7, 2000 at the Hotel Jerome in Aspen, Colorado. Beginning with the first meeting in 1985, these conferences have served as the catalysts for many of the fundamental discoveries that have contributed major insights into these important diseases of the nervous system. These meetings provide a regular forum to bring together basic researchers, geneticists, clinicians, and others familiar with NF1 and NF2 to share their latest research findings and experiences. NNFF Consortia have been instrumental in the rapid advances in the molecular understanding of NF1 and NF2, including the cloning of the responsible genes, establishment of diagnostic tests, exploration of protein function, development of animal models, and creation of a network of patients and clinicians to facilitate clinical trials. The 2000 Consortium meeting has been organized as an open meeting that will be advertised to the general scientific community. Each of six platform sessions will be chaired by an expert in the field. These sessions will include 2 invited speakers from the NF research community with the other presentations to be selected from submitted abstracts. Participation by junior investigators will be encouraged and the organizers anticipate, on basis of previous Consortium meetings, that a substantial percentage of the speakers will be young investigators. Speakers will be instructed to limit their presentations to approximately half of the allotted time, with the remainder dedicated to open discussion among all participants. In addition, longer talks by four keynote speakers who are leaders in scientific fields or emerging technologies that are directly relevant to NF research will be incorporated into some of the scientific sessions. Two poster sessions will also be held which will allow investigators to present abstracts that are not selected for one of the oral sessions. In addition to updating investigators working on NF1 and NF2 on the latest research developments, this meeting will help to identify critical gaps in our knowledge as well as strategies and collaborations to address them. An improved understanding of the molecular basis of NF1 and NF2 will play an essential role in developing improved therapies for the complications of these disorders and has broad implications to the fields of developmental neurobiology and cancer research.

DESCRIPTION: (provided by applicant) Children with the common inherited disorder neurofibromatosis, type 1 (NF1) are predisposed to myeloid leukemia, particularly juvenile chronic myelomonocytic leukemia (JMML). The NF1 gene (NF1) encodes a GTPase activating protein (GAP) called neurofibromin that stimulates GTP hydrolysis on the p21ras (Ras) family of signaling proteins. We have shown that NF1 functions as a tumor suppressor gene in myeloid cells by negatively regulating Ras. In the current period of support, we have exploited a murine model to investigate mechanistic questions related to the role of NF1 in myeloid growth control and to perform preclinical studies of rational therapeutics. These studies strongly implicate the growth factor GM-CSF as playing a central role in the aberrant growth of murine NF1 mutant cells and in human JMML. In the competing renewal of this translational research project, we will extend these studies using expertise and reagents developed during the past 4 years. This application has 4 specific aims. The experiments proposed under aim 1 involve detailed mechanistic studies of the effects of NF1 inactivation on signal transduction, apoptosis, and cell cycle control in myeloid lineage cells. We will also take a genetic approach to test the role of GM-C SF signaling in a myeloproliferative disorder (MPD) that arises in JunB mutant mice. In aim 2, we propose studies to elucidate the role of GM-CSF in fetal liver cell engraftment that might be relevant to the pathogenesis of JMML. Our third aim proposes a combination of in vivo and in vitro approaches to contrast the effects of expressing oncogenic Ras and inactivating NF1 in myeloid cells. These studies will exploit a novel mouse model developed by our collaborator Tyler Jacks. In aim 4, we examine how the adapter molecule p62DOK regulates myeloid growth by interacting with the Ras GTPase activating protein p12OGAP. Together, these studies will provide new insights into how Ras signaling is normally regulated in myeloid cells, and how hyperactive Ras contributes to leukemogenesis.

Myeloid malignancies are characterized by transformation in the stem cell compartment with clonal outgrowth of progeny that demonstrate considerable variability with respect to the degree of differentiation, apoptosis, and blast proliferation. Extensive experimental data implicate aberrant signal transduction as playing a fundamental role in leukemic growth. Mutant tyrosine kinases that contribute to myeloid leukemogenesis such as Flt3 and the BCR-ABL represent excellent targets for the development of molecular therapeutics. Although protein tyrosine phosphatases play an essential role in controlling kinase signaling networks, the role of these proteins in cancer pathogenesis has received limited attention. In studies supported by this project, we identified somatic mutations in the PTPN11 gene in ~35% of patients with juvenile myelomonocytic leukemia (JMML) and in ~5% of acute myeloid leukemias. PTPN11 encodes SHP-2, a non-receptor tyrosine phosphatase that relays signals from many activated growth factor receptors to Ras and other effectors. In other studies, we showed that leukemia-associated PTPN11 alleles encode gain-of-function SHP-2 proteins that induce aberrant hematopoietic progenitor colony growth. PTPN11 is thus the first human oncogene encoding a protein tyrosine phosphatase. Murine Ptpn11 mutant embryos succumb in utero with hematopoietic defects. We found that oncogenic KrasG12D does not rescue this phenotype, and unexpectedly discovered that SHP-2 phosphatase activity in not required for normal hematopoiesis. Germline PTPN11 mutations are also the predominant cause of Noonan Syndrome (NS), a common developmental disorder characterized by skeletal, cardiac, and hematologic abnormalities. Children with NS are at increased risk of developing JMML. In the course of investigating an unusual child with JMML, we unexpectedly identified germline KRAS mutations as a cause of NS. We have extensively characterized these mutant alleles, which encode amino acid substitutions not found in cancer. These observations have contributed substantially to the emerging paradigm that hyperactive Ras signaling through the Raf/MEK/ERK effector cascade plays a fundamental role in both development and cancer. We will use the additional two years of support to pursue the following research goals: (1) to perform mechanistic studies to elucidate how SHP-2 and Ras interact in hematopoiesis and myeloid growth control;and (2) to model germline KRAS mutations that cause human developmental disorders in the mouse and to use the novel strains to begin characterizing their phenotypic and biochemical consequences.

[unreadable] DESCRIPTION (provided by applicant): Our Medical Scientist Training Program (MSTP), entering its 28th Year July I, 2006, is designed to provide trainees with specialized preparation for research-oriented careers in academic medicine. MSTP offers rigorous training in both basic and clinical sciences leading to the combined M.D., Ph.D. degrees. Twelve trainees with outstanding credentials and research backgrounds are recruited each year from a national pool of applicants. Admission to the MSTP is highly competitive and UCSF has been very successful in attracting students over the past 27 years. There are currently 68 trainees and 94 graduates. The general features of the training program are summer laboratory rotations, two years of preclinical medical school coursework integrated with one or two core graduate courses followed by either a clinical clerkship or another laboratory rotation. The trainee then completes three to four years of graduate courses and research, and a final 18-24 months of clinical training. During the first two years, trainees attend a weekly MSTP course designed to provide exposure to research faculty at UCSF. During their graduate research years, the trainees participate in an MSTP preceptorship designed to maintain their clinical skills. Trainees select Ph.D. thesis advisors from a pool of internationally recognized investigators. The program is administered and trainee progress is monitored by an Advisory Council composed of clinical and basic science faculty with outstanding research records who maintain a strong commitment to our program. A major goal of the MSTP is to integrate clinical and basic research training without compromising the quality of either degree. [unreadable] [unreadable] This training program is designed to optimally train physicians for careers in biomedical research. By optimally preparing such individuals for such integrated careers, we will establish a group of physician scientists who will contribute to more rapid progress in medical research and will be in a better position translate of such research towards the better diagnosis, management and treatment of human disease. [unreadable] [unreadable] [unreadable]

The overall goal of the Hematopoietic Malignancies Program is to improve the care of patients with leukemia and lymphoma through focused research efforts. Three principles underlie this multi-disciplinary program: (1) investigating how normal hematopoietic cells regulate growth, differentiation and death are integral to understanding how these processes are perturbed in cancer; (2) laboratory studies of primary leukemia and lymphoma specimens can provide complementary insights into mechanisms of hematopoietic cell growth and leukemogenesis; and, (3) translating research insights into innovative preclinical and clinical trials is integral to improving the care or patients afflicted with cancer. Applying this philosophy to the problems of leukemia and lymphoma has resulted in a Program that includes a diverse and highly interactive group of clinical, translational, population sciences, and basic investigators who utilize a variety of experimental methods to conduct studies in normal and malignant hematopoiesis. The Program takes advantage of exceptional institutional strengths in basic sciences, outstanding clinical programs for the care of adults and children with leukemia and lymphoma, epidemiology, and a tradition of cross-disciplinary collaboration. During the current period of support, Program Members collaborated extensively to contribute novel data to conduct translational, clinical, and epidemiologic studies in hematologic cancers, to model these diseases in the mouse, and to characterize the biochemical consequences of leukemia and lymphoma-associated mutations. Senior Members have effectively mentored junior faculty, and a number of talented young investigators have joined the Program. Monthly meeting have facilitated productive collaborations. Dr. Kevin Shannon, the Program Leader, is a physician-scientist who is active in patient care, clinical investigation, and laboratory research. Dr. Charles Linker, the Program Co-Leader, is a national leader in developing new therapies for leukemia and lymphoma. The goals of the Program are enhanced by Core facilities provided by the Cancer Center and.by interactions with other Center Programs. The Program has $7,850,069 Total peer reviewed support for the last budget year. The Program has 16% intra-programmatic and 26% interprogrammatic publications.

ABSTRACT This Ruth L. Kirschstein National Research Service Award Postdoctoral Research Training Grant renewal application is submitted in response to Program Announcement PA-16-152 for applications to provide institutional T32 awards to train young researchers who will become independent investigators conduting research that addresses important problems in human health. The application requests continued resources to support four physician/scientists each year with MD or MD/PhD degrees who receive mentored research that focuses on the etiology, pathogenesis, and treatment of pediatric malignancies. The overall objective of this T32 program is to train new investigators who will improve the care of children with cancer. The rationale for the program is based on the substantial burden of pediatric cancer in the United States and the well- documented and critical need to provide mentored career development support for young physicians that will enable them to become fully independent and productive laboratory researchers. As such, they will be poised to improve the health of children with cancer throughout the world by bringing state-of-the-art expertise to bear on problems such as inherited predispostions, environmental factors, the toxic and relatively non-specific nature of current therapies, and long-term adverse effects of mutagenic treatments. The design of this program involves harnessing the expertise of world-class research scientists who serve as mentors for interdisiciplinary training. We have successfully implemented distinct trainng tracks for laboratory-based and clincial/translational investigators. We believe that the progress of this T32 program since its inception in 2007 demonstrates tht UCSF has the vision, experience, and infrastructure to train the next generation of leaders in childhood cancer research. In this renewal application, we provide evidence that the Department or Pediatrics and Division of Hematology/Oncology together with the broader UCSF research community comprise an exceptional environment for preparing young physicians for productive careers as independent investigators the field of childhood cancer. This application describe a comprehensive plan for idnetifying, training, and mentoring these individuals. This application is directly relevant to human health as it requests funds to support mentored career development for outstanding young physician/investigators who will become independent researchers in the field of childhood cancer. Programs like this one represent an investment in the future of public health as the researchers who are trained through this award will be equipped to harness state-of-the-art research techniques to attack a formidable health problem in the pediatric population. The long-term outcome of intensive and innovative training provided by this T32 award will be effective new treatments for childhood cancers.

Aberrant signal transduction is a fundamental mechanism underlying malignant growth. The Ras family of signal switch proteins is deregulated in myeloid malignancies by genetic mechanisms that include NRAS and KRAS2 point mutations, the BCR-ABL fusion, PTPN11 mutations, and NF1 inactivation. However, with the notable exception of imatinib mesylate, limited progress has been made toward achieving the goal of developing effective and safe inhibitors of signaling molecules that promote aberrant growth. A major reason for this is that drug discovery is ultimately a biochemical problem that extends beyond our current genetic understanding of cancer biology. A particular bottleneck has been the failure of immortalized cell lines to accurately mimic the behavior of the deregulated signaling networks found in primary cancer cells. In previous studies supported by this award, we showed that the NF1 gene, which encodes a GTPase activating protein (GAP) for Ras, functions as a tumor suppressor gene in immature myeloid cells by negatively regulating Ras signaling. We also exploited a "first generation" mouse model of myeloproliferative disease (MPD) that results from transplanting homozygous Nf1 mutant liver cells into irradiated recipient mice to demonstrate that GM-CSF plays a central role in the abnormal proliferation of Nf1-deficient hematopoietic cells, and to perform preclinical studies of an inhibitor of the Ras processing enzyme farnesyltransferase. In the current period of support, we developed strains of mice that accurately model the genetic and biochemical consequences of hyperactive Ras in primary hematopoietic cells by using the Mx1- Cre strain to ablate a conditional mutant Nf1 allele or to activate oncogenic Kras expression from its endogenous promoter. The goals of this renewal application are to characterize how leukemia-associated genetic lesions deregulate Ras-regulated signaling networks, to identify targets for therapeutic intervention, to use these tractable new models as platforms for testing novel agents, and to identify genes and pathways that cooperate with hyperactive Ras in myeloid leukemogenesis. We propose three specific aims: (1) To characterize and compare the biochemical consequences of oncogenic KrasG12D expression and Nf1 inactivation by interrogating a network of signaling molecules in primary bone marrow cells and in defined subsets of stem/progenitor cells. (2) To investigate the contributions of specific Ras effectors to aberrant growth by expressing mutant proteins in primary hematopoietic cells and by generating and analyzing novel strains of "knock in" mice. (3) To utilize retroviral insertional mutagenesis as a general strategy for uncovering genes that cooperate with oncogenic Kras to induce progression from MPD to AML, and to test how these mutations modulate therapeutic responses in vitro and in vivo.

Loss of chromosome 7 (monosomy 7) and deletion of a segment of the long arm [del(7q)] are recurring cytogenetic abnormalities in de novo and therapy-related myeloid malignancies that are associated with a poor prognosis. In previous studies supported by this award, cytogenetic analysis delineated a commonly-deleted segment (CDS) in patients with myeloid disorders characterized by a del(7q) within band q22 that accounts for most cases, and a second CDS in bands q32-34. Using an ordered set of yeast artificial chromosome clones as probes, fluorescence in situ hybridization experiments were then performed on leukemias with deletion breakpoints within 7q22. These studies implicated a ~2.5 Mb CDS as harboring a tumor suppressor gene (TSG) that is inactivated in myeloid malignancies. During this period of support, we have extensively characterized this CDS, identified and cloned 19 known and novel genes from the interval, analyzed leukemia samples for mutations in these candidate TSGs, and performed Taqman real time quantitative polymerase chain reaction experiments to measure expression levels in normal and leukemic human bone marrows. These studies did not uncover either pathogenic mutations or a consistent reduction in expression levels in any candidate TSG. We also harnessed chromosome engineering technology to introduce loxP sites flanking a ~2 Mb syntenic interval on mouse chromosome 5, and have generated conditional and germline A5 mice. We are currently pursuing a number of strategies to identify candidate TSGs and cooperating genes in these strains. Our underlying hypotheses are: (1) that a myeloid TSG resides either within this 7q22 CDS or in a DNA segment proximal to this CDS;and, (2) that this gene is either inactivated or demonstrates reduced expression in myeloid malignancies with monosomy 7 or by a del(7q). We will exploit reagents that we have generated and a diverse collection of leukemia samples to pursue this hypothesis through two aims: (1) to use heterozygous and homozygous A5 mice to identify candidate human 7q22 TSGs and other genes that cooperate with the A5 deletion in leukemogenesis;and (2) to integrate new data developed by Projects 2 and 3 and by other researchers to prioritize candidate myeloid TSGs in the interval immediately proximal to the 7q22 CDS that might cooperate with loss of the 7q22 CDS. We will interrogate human leukemia specimens for mutations in genes located in a 2.1 Mb interval immediately proximal to the current CDS, and develop reagents for modeling loss of this more proximal segment in the mouse. Project 4 is highly interactive with Projects 2 and 3;together, these projects seek to identify the spectrum of genetic mutations, and genetic pathways leading to alkylating-agent induced t-AML.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. NOTCH1 encodes a transmembrane receptor that is critical for T cell development and is mutated in ~55% of human T lineage acute lymphoblastic leukemia (T-ALL) cases. Activation of the Notch1 protein requires cleavage by the gamma-secretase protease, which releases the Notch1 intracellular domain (NICD) that then translocates to the nucleus and upregulates transcription. Mutated forms of Notch1 are gamma-secretase dependent and several gamma-secretase inhibitors (GSIs) have been developed. We harnessed retroviral insertional mutagenesis (RIM) in Mx1-cre;LSL-KrasG12D mice to generate a large panel of genetically and biochemically diverse T-ALLs. In this model, Notch1 is frequently mutated and NICD can be detected using a specific antibody that recognizes Val 1477 that is exposed only after gamma-secretase cleavage. We have thoroughly interrogated Notch1 dependence in 20 tumor-derived cell lines and found that sensitivity to GSI correlates with the presence of NICD and GSI treatment reduces NCID levels. This work is currently in press at PNAS. Interestingly, we identified 3 cell lines that robustly express a protein recognized by the NICD specific antibody, but are GSI-resistant. This protein does not disappear following GSI exposure. Further analysis revealed that these lines do not express Notch1 mRNA or have upregulation of Notch1 target genes, indicating that Notch1 is not activated. Importantly, we never detect this protein and would like to use mass spectrometry to determine its identity. Future experiments would involve 1) using shRNA to knockdown the protein in T-ALL cells to assess its role in tumorigenesis, and 2) screening human T-ALL patient samples for mutations in the gene that encodes this protein.

The p21[ras] (Ras) family of signal switch proteins is deregulated in myeloid malignancies by genetic mechanisms that include NRAS and KF^S2 point mutations, the BCR-ABL fusion, PTPN11 mutations, and NF1 inactivation. We developed accurate mouse models of myeloproliferative disease (MPD) by exploiting the Mx1-Cre transgene to ablate a conditional mutant Nf1 allele or to activate oncogenic Kras[G121D] expression from its endogenous promoter (1, 2). In recent work, we exploited this strategy to induce endogenous oncogenic Nras[G12D] expression in hematopoietic cells, and unexpectedly observed marked phenotypic differences in Kras and Nras mutant mice. Studies in this new model formed the basis of a successful application for supplemental funding support through the ARRA to extend the scope of this R37 award to investigate leukemogenesis in Nras mice. We have made extensive use of retroviral insertional mutagenesis to induce progression from MPD to acute myeloid leukemia (AML) and T lineage acute lymphoblastic leukemia (T-ALL) in Nf1, Kras, and Nras mutant mice, and we are harnessing these aggressive and genetically heterogeneous cancers to investigate mechanisms of drug response and resistance in vivo.

DESCRIPTION (provided by applicant): RAS proto-oncogenes are mutated at high frequency in many different malignancies. Thus, developing effective therapeutic strategies for reversing the biochemical consequences of oncogenic Ras is a fundamental obstacle to reducing the worldwide burden of cancer. Although oncogenic RAS alleles encode gain-of-function proteins that are robustly expressed in cancer cells, intrinsic characteristics of the Ras/GTPase activating protein (Ras/GAP) molecular switch pose difficult, if not insurmountable, challenges to developing targeted inhibitors. The undruggable biochemical properties of the oncogenic Ras/GAP switch represent a central unsolved problem in cancer therapeutics. Activated Ras engages a complex network of kinase effector cascades of which the Raf/MEK/ERK and phosphoinositide-3-OH kinase (PI3K), Akt, mammalian Target of Rapamycin (PI3K/Akt/mTOR) pathways are strongly implicated in cancer initiation and maintenance. In this project, we will exploit transplantable primary myeloid and lymphoid leukemias from strains of Nf1 mutant and Kras/Nras knock in mice that accurately model human cancers as a controlled evolutionary system and experimental platform for interrogating responses to small-molecule inhibitors. In particular, we have transplanted ~40 primary leukemias into cohorts of mice, and have treated these recipients with MEK and PI3K inhibitors alone and in combination. We have isolated multiple, independent drug resistant leukemias from these controlled preclinical trials, and have shown that acquired resistance follows distinct evolutionary trajectories. These data recapitulate, with remarkable fidelity, the initial response and ultimate relapse of advanced human cancers treated with targeted inhibitors. This general approach has the additional advantage of providing a tractable forward genetic system for discovering and validating mechanisms of de novo and acquired resistance. Here we propose to use these novel reagents to interrogate in vivo clonal selection of cancers driven by oncogenic Ras signaling in response to treatment with MEK and PI3K inhibitors as well as mechanisms of response and resistance. The specific aims of this PQ proposal are: (1) to investigate the evolution of acquired resistance to MEK inhibitors in primary AML characterized by Nf1 inactivation or by oncogenic Nras/Kras mutations; and (2) to elucidate the clonal architecture, evolution, and drug responses in T-ALLs from wild-type and Kras mutant mice. Our overall goals are: (1) to reveal biologic principles underlying how the selective pressure imposed by MEK and/or PI3K inhibitor treatment leads to clonal evolution of Ras-driven cancers in vivo; (2) to discover specific genes and pathways that confer resistance to targeted anti-cancer agents; and, (3) to use these data to develop therapeutic paradigms for reversing the adverse biochemical outputs of oncogenic Ras that can be translated through human clinical trials.

ABSTRACT ? DEVELOPMENTAL RESEARCH PROGRAM The Developmental Hyperactive Ras Tumor (DHART) SPORE Developmental Research Program (DRP) will support innovative pilot projects to advance the diagnosis and treatment of early stage tumors and advanced cancers characterized by germ line and somatic NF1 mutations. The DRP will accomplish this goal by supporting rigorous translational research in the areas of population science, therapeutics, and mechanisms of disease. We expect that many investigators will utilize DRP support to generate preliminary data leading to successful grant applications to the NIH, the Department of Defense NF Research Program, and other funding agencies, thereby advancing research activities related to NF1-assocated tumors. The translational studies supported by the DRP will also have the potential to replace SPORE projects that have been completed or are not progressing satisfactorily toward achieving their objectives. One objective of the DRP is to fund established investigators who seek to develop a new research theme in the field of NF1-related cancers. This SPORE also includes a Career Development Program (CDP), which is described in a separate section of this application that has the complimentary goal of supporting junior investigators who wish to develop innovative research projects related to the goals of this SPORE. The institutions participating in this application have committed a total of $1.3 million to support the Developmental Programs component of the DHART SPORE (DRP and CDP). Investigators from any of these centers can apply for DRP funds, and we will also consider proposals from researchers at other institutions. The specific aims of the DRP are: (1) To support the development of new techniques and resources to advance translational research to improve the treatment of NF1-associated tumors; (2) to provide funds that will allow investigators to generate pilot feasibility data for grant applications to the NIH or other federal funding agencies; (3) to expand the scope of research in NF1-assocaited tumors with an emphasis on supporting high risk/high reward projects; and (4) to recruit talented investigators into NF1 research as it relates to cancer, and to promote new collaborations in this field through structured interactions with DHART SPORE investigator and assistance from the Administrative, Omics, and Biospecimen/Pathology Cores. The DRP will provide evaluation, funding, and oversight for a minimum of two pilot projects per year, with the potential for a single renewal. Each project will receive a minimum of $50,000 with a maximum of $75,000. Applications for DRP support (including requests for a second year of funding) will be evaluated through a competitive process with review by a Developmental Programs Steering Committee comprised on senor scientists participating in this SPORE and oversight and guidance from the External and Internal Advisory Boards.

DESCRIPTION (provided by applicant): With a worldwide incidence of 1 in 3000, neurofibromatosis type 1 (NF1) is the most common inherited cancer predisposition syndrome. NF1 is caused by germ line mutations in the NF1 tumor suppressor gene (TSG), which encodes a GTPase activating protein (GAP) called neurofibromin that forms a molecular complex with activated Ras-GTP and negatively regulates Ras signaling by accelerating GTP hydrolysis. NF1, the most common rasopathy, has a propensity to develop neoplastic diseases that progress to aggressive cancers and frequently affect children, adolescents, and young adults. A common feature of NF1-associated neoplasms and malignant tumors is somatic loss of the normal NF1 allele. Importantly, limited epidemiologic data support the hypothesis that patients with NF1 who are cured of a primary cancer are at increased risk of developing treatment-induced secondary neoplasms (SNs). Together, the neoplastic diseases and aggressive malignancies that develop in NF1 are a substantial cause of morbidity and premature mortality. In addition to its role as an initiating mutation in NF1-associated cancers, recent genome-wide sequencing studies uncovered frequent somatic NF1 mutations in glioblastoma, acute myeloid leukemia, adenocarcinoma of the lung, and other sporadic cancers. Importantly, there are currently no mechanism-based therapies for cancers with mutations in genes encoding components of the Ras/GAP molecular switch (KRAS, NRAS, HRAS, and NF1). Thus, our focus will be developing effective higher-quality healthcare delivery options for children, adolescents and young adults with neurofibromatosis (NF) and provide insights that will benefit the entire Ras community. The overall goal of this Developmental and Hyperactive Ras Tumor (DHART) SPORE is to implement effective targeted molecular therapies for neoplasms and cancers by conducting integrated, mechanistically based translational research. The overarching objectives of this highly-qualified, collaborative group are : 1) to evaluate novel therapeutics in validated preclinical models and in the treatment of patients with NF1; 2) to identify risk factors of individuals with NF1 to acquire spontaneous and treatment-associated second malignancies; and 3) to decrease tumor associated morbidity and mortality of patients with NF1. This application draws on the expertise of an accomplished team of clinical investigators, physician/ scientists, and basic researchers with an extensive track record of productive collaborations. This program encompasses four highly integrated projects and three cores. The theme of translational therapeutics informs Projects 1 through 3, and the focus of project 4 exemplifies the role of cancer epidemiology and prevention. State-of-the-art core facilities will inform the patient-oriented cancer therapeutic and prevention aspects of this SPORE by elucidating mutations that contribute to of cancer pathogenesis and by defining biomarkers of drug response and resistance that will inform next generation treatments.

ABSTRACT ? PROJECT 3 Children with neurofibromatosis type 1 (NF1) are predisposed to juvenile myelomonocytic leukemia (JMML), an aggressive myeloproliferative neoplasm (MPN). The median survival of JMML patients is <1 year without hematopoietic stem cell transplantation (HSCT), and the overall cure rate is ~50% after HSCT. Our studies showing that NF1 functions as a tumor suppressor gene in JMML patients implicated hyperactive Ras signaling in the pathogenesis of this aggressive cancer. Consistent with this hypothesis, subsequent studies uncovered mutations in the NRAS, KRAS, PTPN11, and CBL genes in JMML patients. Despite the routine use of HSCT in JMML, up to 30% of patients progress to acute myeloid leukemia (AML). Consistent with the molecular genetics of JMML, using the Mx1-Cre transgene to inactivate a conditional mutant Nf1flox allele in the hematopoietic compartment induces a JMML-like MPN is induced in mice, and our preclinical studies in this genetically accurate mouse model revealed remarkable efficacy of potent and selective MEK inhibitors. Interestingly, we found that treatment did not eradicate mutant cells, but modulated their proliferation and differentiation in vivo. Based on these studies, Project 3 of this DHART SPORE will pursue two specific aims. First, we will conduct an Investigator-initiated trial of the FDA-approved MEK inhibitor trametinib in relapsed and newly diagnosed JMML. We will also interrogate molecular mechanisms of response and resistance through the use of sensitive residual disease assays that harness next-generation sequencing technologies to monitor mutant allele burden in JMML specimens. We hypothesize that MEK inhibition will markedly reduce or eradicate JMML cells in a subset of children with JMML, and will induce clinical improvement without altering mutant allele frequency in others. We further postulate that a complete genetic response will predict a favorable outcome, and we will interrogate leukemia cells from patients who initially respond to trametinib and then relapse to identify candidate resistance mutations. In Aim 2, we will investigate human JMML cells and use mouse models of MPN and AML characterized by Nf1 inactivation to investigate how secondary mutations identified in JMML specimens influence the response to trametinib, and to functionally validate candidate mechanisms of drug resistance. Project 3 will benefit from and inform the other Projects in this SPORE, and are dependent on the Administrative, Omics, and Biospecimens/Pathology Cores for successfully achieving its goals.

? DESCRIPTION (provided by applicant): RAS proto-oncogenes are mutated in ~30% of human cancers, but no mechanism-based treatments exist for reversing the biochemical output of oncogenic Ras proteins, which are exceedingly difficult targets for rational drug discovery. Our long-term goal is to implement mechanistic strategies to selectively inhibit the growth of cancers with somatic RAS mutations. In this project, we will investigate the Ras palmitoylation/depalmitoylation cycle, which regulates the subcellular trafficking of the N-Ras, H-Ras, and K-Ras4a isoforms, as a therapeutic target for selectively inhibiting the growth of malignancies with oncogenic NRAS mutations. Acyl protein thioesterase 1 and 2 (APT1 and APT2) catalyze N-Ras depalmitoylation and we found that first generation chemical inhibitors designed to inhibit these enzymes (Palmostatin B and Palmostatin M) selectively reduced the growth of primary hematopoietic progenitors and leukemia cells expressing oncogenic N-RasG12D. However, our recent studies also infer the existence of additional biochemical targets of these compounds that are essential for the growth of NRAS mutant cancer cells. This project involves a cross-disciplinary collaboration that brings together investigators with extensive expertise in synthetic chemistry (Dr. Howell), enzymology and chemical biology (Dr. Cravatt), and hematologic cancer, Ras signaling, and preclinical therapeutics (Dr. Shannon). We have collaborated to generate extensive preliminary data and novel reagents, which we will use to pursue the goals of: (1) identifying additional biochemical targets of the palmostatins; (2) developing new chemical inhibitors with improved potency and selectivity for palmostatin targets; (3) using these inhibitors combined with genetic methods to discern the relevant enzyme(s) that regulate N-Ras depalmitoylation in cancer cells; and, (4) utilizing human cancer cell lines and a new strain of mice to interrogate the palmitoylation/depalmitoylation cycle as a therapeutic target in early stage and advanced NRAS-mutant cancers. We will address these questions through two highly integrated specific aims. In Aim 1, we will identify additional biochemical targets of palmostatin M, design and characterize new chemical inhibitors, and evaluate the efficacy of these compounds in cancer cells with NRAS mutations. In Aim 2, we will utilize a novel strain of NrasG12D,C181S knock in mice to ask if the palmitoylation/depalmitoylation cycle is required for the growth of oncogenic Nras-driven cancers in vivo. These studies will rigorously assess the importance of the palmitoylation/depalmitoylation cycle and inform the development of new therapeutic strategies for cancers with oncogenic RAS mutations.

ABSTRACT ? CAREER DEVELOPMENT PROGRAM The Developmental Hyperactive Ras Tumor (DHART) SPORE Career Development Program (CDP) will support faculty at the Assistant Professor level to engage in translational research focused on NF1 and cancer. The specific purpose or the CDP is to advance the careers of junior investigators who are developing innovative research projects related to the goal of implementing more effective and less toxic therapies for cancers characterized by NF1 mutations. The DHART SPORE also includes a Developmental Research Program (DRP), which has the complementary mission of funding established investigators who seek to develop new research related to NF1-related cancers and is presented in a separate section of this application. The multi-institutional nature of this SPORE leverages a large and talented pool of junior faculty from multiple disciplines. Each institution views the CDP as a vehicle for recruiting, training, and advancing young scientists working in the area of translational cancer research related to NF1, and they have committed $1.3 million to support the Developmental Programs component of the DHART SPORE (DRP and CDP). The Specific Aims of the CDP are: (1) to support new investigators as they develop innovative research programs with the goal of contributing new knowledge to the field of NF1-related cancer research while generating preliminary data that will allow them to successfully apply for independent extramural funding; (2) to provide young researchers with didactic training that will equip them with essential skills and knowledge for success as an independent investigator in the field of NF1-related cancer research; (3) to mentor young translational researchers through regularly scheduled one-on-one meetings with a designated senior faculty member and a structured Individual Career Development Plan (IDP) who will fully integrate the CDP-supported investigator into the fabric of the DHART SPORE where they have access to advice, resources, and potential collaborators; and, (4) to encourage the recruitment of ethnic minorities, those from disadvantaged backgrounds, women and lesbian/gay/bisexual/transgender (LGBT) populations into research focused on NF1-associated cancers. The DHART SPORE will link vertically and horizontally to diversity programs at all of the consortium institutions. Investigators from any of these centers can apply for CDP funds, and we will also consider proposals from researchers at other institutions. The CDP will fund a minimum of two projects per year at an annual budget of $50,000 - $75,000. Applications for CDP support (including requests for a second year of funding) will be evaluated through a competitive process with review by a Developmental Programs Steering Committee comprised on senor scientists participating in this SPORE and oversight and guidance from the External and Internal Advisory Boards.

ABSTRACT Recurring losses of large chromosomal regions are a hallmark of pediatric and adult cancer genomes that pose exceptional challenges for uncovering how these deletions contribute to malignant growth. Monosomy 7 (-7) and del(7q) (-7/del(7q)) are recurring cytogenetic abnormalities in de novo myeloid malignancies that are strongly associated with cases of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) arising in children and in adolescent/young adult (AYA) patients with inherited cancer predispositions and in those who develop myeloid malignancies after treatment for a primary cancer. Therapy-induced MDS and AML (t-MDS and t-AML) are particularly relevant to the pediatric and AYA population due to modern intensive treatment protocols for many solid cancers, which are frequently curative. As a result, there is a large and growing population of ?at risk? pediatric and AYA cancer survivors. Unfortunately, t-MDS/t-AML and other myeloid malignancies with chromosome 7 deletions are highly refractory to current therapies. Extensive cytogenetic and genome wide analysis studies implicate deletions of chromosome band 7q22 in leukemogenesis; however, sequencing studies and transcriptome analysis did not reveal frequent homozygous inactivation of any candidate 7q tumor suppressor gene in myeloid malignancies. These data implicate haploinsufficiency for one or more 7q genes in leukemogenesis, which pose formidable challenges for elucidating the underlying molecular mechanisms. To address this fundamental problem, we deployed chromosome engineering to create 5A3+/del and 5G2+/del mice, which respectively harbor deletions in mouse chromosome bands 5A3 and 5G2. These deletions span ~4 MB of genomic DNA that is syntenic to the most common 7q22 deletions identified in human patients. 5A3+/del hematopoietic stem and progenitor cells (HSPC) exhibit ?preleukemic? abnormalities, but these mice do not spontaneously develop MDS or AML. Preliminary studies of 5G2+/del mice also revealed HSPC abnormalities and exposing this strain to N-ethyl-N-nitrosourea (ENU) accelerated the development of hematologic cancer. We will utilize these novel models of 7q22 deletions to pursue the following specific aims: (1) to functionally interrogate hematopoiesis in 5G2+/del and 5A3+/del/5G2+/del mice and to observe cohorts of mice for the development of myeloid malignancies; (2) to investigate the effects of DNA damaging agents on 5A3+/del/5G2+/del HSPC; and, (3) to model the complex genetics of myeloid malignancies with -7/del(7q) by introducing cooperating mutations into 5A3+/del/5G2+/del HSPC and assessing the phenotypic and functional consequences in vivo. Genetically engineered mice that accurately model recurrent chromosome band 7q22 deletions found in human myeloid malignancies are a versatile system for performing functional studies and testing new therapeutic strategies.