Search

Despite decades of clinical use, mechanisms of glucocorticoid resistance are poorly understood. We treated primary murine T lineage acute lymphoblastic leukemias (T-ALLs) with the glucocorticoid dexamethasone (DEX) alone and in combination with the pan-PI3 kinase inhibitor GDC-0941 and observed a robust response to DEX that was modestly enhanced by GDC-0941. Continuous in vivo treatment invariably resulted in outgrowth of drug-resistant clones, ~30% of which showed low glucocorticoid receptor (GR) protein expression. A similar proportion of relapsed human T-ALLs also exhibited markedly reduced GR protein levels. De novo or pre-existing mutations in the gene encoding GR (Nr3c1) occurred in relapsed clones derived from multiple independent parental leukemias. CRISPR/Cas9 gene editing confirmed that loss of GR expression confers DEX resistance. Exposing drug-sensitive T-ALLs to DEX in vivo altered transcript levels of multiple genes, and this response was attenuated in relapsed T-ALLs. These data implicate reduced GR protein expression as a frequent cause of glucocorticoid resistance in T-ALL.

Oncogenic RAS mutations pose substantial challenges for rational drug discovery. Sequence variations within the hypervariable region of Ras isoforms underlie differential posttranslational modification and subcellular trafficking, potentially resulting in selective vulnerabilities. Specifically, inhibiting the palmitoylation/depalmitoylation cycle is an appealing strategy for treating NRAS mutant cancers, particularly as normal tissues would retain K-Ras4b function for physiologic signaling. The role of endogenous N-RasG12D palmitoylation in signal transduction, hematopoietic differentiation, and myeloid transformation is unknown, and addressing these key questions will inform efforts to develop mechanism-based therapies. To evaluate the palmitoylation/depalmitoylation cycle as a candidate drug target in an in vivo disease-relevant model system, we introduced a C181S mutation into a conditional NrasG12D “knock-in” allele. The C181S second-site amino acid substitution abrogated myeloid transformation by NrasG12D, which was associated with mislocalization of the nonpalmitoylated N-Ras mutant protein, reduced Raf/MEK/ERK signaling, and alterations in hematopoietic stem and progenitor populations. Furthermore, hematologic malignancies arising in NrasG12D/G12D,C181S compound heterozygous mice invariably acquired revertant mutations that restored cysteine 181. Together, these studies validate the palmitoylation cycle as a promising therapeutic target in NRAS mutant cancers.

The lack of predictive preclinical models is a fundamental barrier to translating knowledge about the molecular pathogenesis of cancer into improved therapies. Insertional mutagenesis (IM) in mice is a robust strategy for generating malignancies that recapitulate the extensive inter- and intra-tumoral genetic heterogeneity found in advanced human cancers. While the central role of "driver" viral insertions in IM models that aberrantly increase the expression of proto-oncogenes or disrupt tumor suppressors has been appreciated for many years, the contributions of cooperating somatic mutations and large chromosomal alterations to tumorigenesis are largely unknown. Integrated genomic studies of T lineage acute lymphoblastic leukemias (T-ALLs) generated by IM in wild-type (WT) and Kras mutant mice reveal frequent point mutations and other recurrent non-insertional genetic alterations that also occur in human T-ALL. These somatic mutations are sensitive and specific markers for defining clonal dynamics and identifying candidate resistance mechanisms in leukemias that relapse after an initial therapeutic response. Primary cancers initiated by IM and resistant clones that emerge during in vivo treatment close key gaps in existing preclinical models, and are robust platforms for investigating the efficacy of new therapies and for elucidating how drug exposure shapes tumor evolution and patterns of resistance., Author summary A lack of predictive cancer models is a major bottleneck for prioritizing new anti-cancer drugs for clinical trials. We comprehensively profiled a panel of primary mouse T lineage leukemias initiated by insertional mutagenesis and found remarkable similarities with human T-ALL in regard to overall mutational burden, the occurrence of specific somatic mutations and large chromosomal alterations, and concordant gene expression signatures. We observed frequent duplication of the Kras oncogene with loss of the normal allele, which has potential therapeutic implications that merit further investigation in human leukemia and in other preclinical models. Mutations identified in mouse leukemias that relapsed after in vivo treatment with signal transduction inhibitors were also observed in relapsed human T-ALL, indicating that this model system can be utilized to investigate strategies for overcoming intrinsic and acquired drug resistance. Finally, preclinical models similar to the one described here that are characterized by a normal endogenous tumor microenvironment and intact immune system will become increasingly important for testing immunotherapy approaches for human cancer.

A dynamic cycle of palmitoylation and depalmitoylation stably localizes N-Ras, but not K-Ras-4b, at the plasma membrane. This selective dependency suggests that targeting palmitoylation could inhibit the growth of NRAS-mutant cancers while leaving normal cells unaffected, due to their intact K-Ras-4b function. We sought to clarify the dependency of oncogenic N-Ras signaling on palmitoylation in a model relevant to human cancer. We used the lox-STOP-lox (LSL) knock-in system (Haigis et al., Nat Genet 2008) to express an NrasG12D oncogene at physiologic levels, and generated a second site mutation abrogating the palmitoylation site (C181S). We then compared Mx1-Cre; NrasLSL-G12D,C181S/LSL-G12D,C181S to Mx1-Cre; NrasWT/WT and Mx1-Cre; NrasLSL-G12D/LSL-G12D (hereafter G12D,C181S, WT, and G12D, respectively). We injected mice with poly(I:C) at weaning to excise the LSL cassette in hematopoietic cells. As reported (Wang et al., Blood 2011; Xu et al., Cancer Disc 2013), G12D mice died early (119-267 days, n=14) from an aggressive myeloproliferative disorder (MPD). In contrast, no mortality was observed in the G12D,C181S and WT cohorts until 18-19 months of age (p=3x10-6 by Kaplan-Meier analysis). Pathologic analysis of 6-month-old mice showed that key features of MPD were fully rescued in the G12D,C181S cohort. Specifically, whereas white blood cell (WBC) counts and spleen sizes were in the normal range in G12D,C181S and WT mice, G12D mice had profound leukocytosis (median WBC count: 52.19 k/μL), splenomegaly (median spleen weight: 0.884 g), and myeloid expansion (n=6). Biochemical analysis revealed that N-RasG12D,C181S accumulated in the GTP-bound conformation, but mostly localized in the cytosol, and failed to hyperactivate the MAPK pathway. We next generated NrasG12D,C181S/G12D compound heterozygous mice to investigate potential interactions between oncogenic NrasG12D alleles with and without the C181S mutation in vivo. Over half of these mice developed hematologic disease and died prematurely, although later than homozygous G12D mutant mice (median survivals: 429 and 225 days; n = 22 and 14, respectively). Common findings in moribund mice were splenomegaly, expansion of myeloid cells, and different degrees of leukocytosis and anemia. Importantly, genetic analysis from diseased mice revealed reduced frequency or absence of the NrasG12D,C181S allele (allelic burden 0-30%) due to secondary somatic genetic events. This observation supports the existence of high selective pressure to overcome growth-suppressive properties of unpalmitoylated N-Ras during leukemogenesis. In conclusion, these studies provide strong genetic evidence validating palmitoylation as a therapeutic target in NRAS mutant hematologic malignancies and establish a novel model for addressing this question in other cancers. Citation Format: Noemi A. Zambetti, Ari J. Firestone, Jasmine C. Wong, Amanda M. Long, Anagha Inguva, Jarrett R. Remsberg, Radu M. Suciu, Benjamin F. Cravatt, Kevin M. Haigis, Kevin Shannon. In vivo evidence validating the palmitoylation/depalmitoylation cycle as a therapeutic target in NRAS mutant hematologic cancers [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A25.

Phosphatidylinositol 3-kinase (PI3K) signaling is essential for RAS-driven transformation. To directly investigate the role of oncogenic K-Ras binding to PI3K in development and tumorigenesis, we generated KrasG12D/+,Y64G/+ mice that express, from the endogenous locus, a K-Ras oncoprotein that also contains a “second site” amino acid substitution at tyrosine 64 (Y64G) that disrupts the interaction between oncogenic K-Ras and PI3K. Surprisingly, KrasG12D,Y64G mice are viable, fertile, and phenotypically unremarkable, although they are born at a lower than expected Mendelian frequency (26% versus the expected 50% on a C57BL/6 strain background). As opposed to the enhanced proliferative rate observed in mouse embryonic fibroblasts (MEFs) from K-RasG12D mice, KrasG12D,Y64G MEFs exhibit wild-type rates of proliferation. Detailed analysis of the hematopoietic compartment in KrasG12D,Y64G mice reveals a reduced proportion of long-term hematopoietic stem cells, but no evidence of the aggressive myeloproliferative neoplasm observed when a conditional mutant KrasG12D is activated in hematopoietic cells. Consistent with these observations, bone marrow cells isolated from K-RasG12D,Y64G mice exhibit a normal pattern of myeloid progenitor colony growth in response to cytokine stimulation. In contrast to the lack of observed hematologic malignancy, 100% of KrasG12D,Y64G mice we examined at 1 year of age showed lung lesions, ultimately succumbing to lung tumors with a median survival of 496 days. Despite the ubiquitous KrasG12D,Y64G expression, these mice survive longer than models with mosaic, adenoviral-Cre recombinase-controlled KrasG12D (median survival of 185 days). The majority of the lung lesions that arise in KrasG12D, Y64G mice are low grade, classified pathologically as atypical lymphoid proliferation or papillary adenomas; a few adenocarcinomas are also observed. These studies reinforce the importance of oncogenic KRas-mediated activation of PI3K for transformation and demonstrate that expressing a Y64G amino acid substitution in the context of oncogenic KrasG12D normalizes cell proliferation, rescues embryonic lethality, abrogates myeloid disease, and attenuates lung tumorigenesis. Beyond the bone marrow and lung, this mutant strain is a potent genetic tool for dissecting the role of aberrant PI3K signaling in pancreatic, colon, and other tissues characterized by tumors driven by somatic KRAS mutations, and also have implications for treating human cancers with KRAS mutations. Citation Format: Jasmine C. Wong, Pedro A. Perez-Mancera, Jangkyung Kim, Kuang-Yu Jen, Scott C. Kogan, Ari J. Firestone, Eric A. Collisson, David A. Tuveson, Kevin Shannon. A second site KrasG12D mutation that impairs PI3K binding rescues embryonic lethality, abrogates myeloproliferative disease, and delays lung tumorigenesis [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A23.

Germline SAMD9 and SAMD9L mutations cause a spectrum of multisystem disorders that carry a markedly increased risk of developing myeloid malignancies with somatic monosomy 7. Here, we describe 16 siblings, the majority of which were phenotypically normal, from 5 families diagnosed with myelodysplasia and leukemia syndrome with monosomy 7 (MLSM7; OMIM 252270) who primarily had onset of hematologic abnormalities during the first decade of life. Molecular analyses uncovered germline SAMD9L (n = 4) or SAMD9 (n = 1) mutations in these families. Affected individuals had a highly variable clinical course that ranged from mild and transient dyspoietic changes in the bone marrow to a rapid progression of myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) with monosomy 7. Expression of these gain-of-function SAMD9 and SAMD9L mutations reduces cell cycle progression, and deep sequencing demonstrated selective pressure favoring the outgrowth of clones that have either lost the mutant allele or acquired revertant mutations. The myeloid malignancies of affected siblings acquired cooperating mutations in genes that are also altered in sporadic cases of AML characterized by monosomy 7. These data have implications for understanding how SAMD9 and SAMD9L mutations contribute to myeloid transformation and for recognizing, counseling, and treating affected families.

NRAS is frequently mutated in hematologic malignancies. We generated Mx1-Cre, Lox-STOP-Lox (LSL)-NrasG12D mice to comprehensively analyze the phenotypic, cellular, and biochemical consequences of endogenous oncogenic Nras expression in hematopoietic cells. Here we show that Mx1-Cre, LSL-NrasG12D mice develop an indolent myeloproliferative disorder but ultimately die of a diverse spectrum of hematologic cancers. Expressing mutant Nras in hematopoietic tissues alters the distribution of hematopoietic stem and progenitor cell populations, and Nras mutant progenitors show distinct responses to cytokine growth factors. Injecting Mx1-Cre, LSL-NrasG12D mice with the MOL4070LTR retrovirus causes acute myeloid leukemia that faithfully recapitulates many aspects of human NRAS-associated leukemias, including cooperation with deregulated Evi1 expression. The disease phenotype in Mx1-Cre, LSL-NrasG12D mice is attenuated compared with Mx1-Cre, LSL-KrasG12D mice, which die of aggressive myeloproliferative disorder by 4 months of age. We found that endogenous KrasG12D expression results in markedly elevated Ras protein expression and Ras-GTP levels in Mac1+ cells, whereas Mx1-Cre, LSL-NrasG12D mice show much lower Ras protein and Ras-GTP levels. Together, these studies establish a robust and tractable system for interrogating the differential properties of oncogenic Ras proteins in primary cells, for identifying candidate cooperating genes, and for testing novel therapeutic strategies.

The leukemogenic effects of Myc drive recurrent trisomy in a mouse model of acute myeloid leukemia., Gain of chromosome 8 is the most common chromosomal gain in human acute myeloid leukemia (AML). It has been hypothesized that gain of the MYC protooncogene is of central importance in trisomy 8, but the experimental data to support this are limited and controversial. In a mouse model of promyelocytic leukemia in which the MRP8 promoter drives expression of the PML-RARA fusion gene in myeloid cells, a Myc allele is gained in approximately two-thirds of cases as a result of trisomy for mouse chromosome 15. We used this model to test the idea that MYC underlies acquisition of trisomy in AML. We used a retroviral vector to drive expression of wild-type, hypermorphic, or hypomorphic MYC in bone marrow that expressed the PML-RARA transgene. MYC retroviruses cooperated in myeloid leukemogenesis and suppressed gain of chromosome 15. When the PML-RARA transgene was expressed in a Myc haploinsufficient background, we observed selection for increased copies of the wild-type Myc allele concomitant with leukemic transformation. In addition, we found that human myeloid leukemias with trisomy 8 have increased MYC. These data show that gain of MYC can contribute to the pathogenic effect of the most common trisomy of human AML.

Monosomy 7 and del(7q) are associated with adverse features in myeloid malignancies. A 2.5-Mb commonly deleted segment (CDS) of chromosome band 7q22 is implicated as harboring a myeloid tumor suppressor gene (TSG); however, molecular analysis of candidate TSGs has not uncovered loss of function. To determine whether haploinsufficiency for the 7q22 CDS contributes to myeloid leukemogenesis, we performed sequential gene targeting to flank a region of orthologous synteny on mouse chromosome band 5A3 with loxP sites. We then generated Mx1-Cre, 5A3fl mutant mice and deleted the targeted interval in vivo. Although excision was inefficient, we confirmed somatic deletion of the 5A3 CDS in the hematopoietic stem cell compartment. Mx1-Cre, 5A3fl mice show normal hematologic parameters and do not spontaneously develop myeloid malignancies. The 5A3fl deletion does not cooperate with oncogenic KrasG12D expression, Nf1 inactivation, or retroviral mutagenesis to accelerate leukemia development and did not modulate responsiveness to antileukemia drugs. These studies demonstrate that it is feasible to somatically delete a large chromosomal segment implicated in tumor suppression in hematopoietic cell populations in vivo; however, our data do not support the hypothesis that the 7q22/5A3 CDS interval contains a myeloid TSG.

Chromosome 7 deletions are highly prevalent in myelodysplastic syndrome (MDS) and likely contribute to aberrant growth through haploinsufficiency. We generated mice with a heterozygous germ line deletion of a 2-Mb interval of chromosome band 5A3 syntenic to a commonly deleted segment of human 7q22 and show that mutant hematopoietic cells exhibit cardinal features of MDS. Specifically, the long-term hematopoietic stem cell (HSC) compartment is expanded in 5A3+/del mice, and the distribution of myeloid progenitors is altered. 5A3+/del HSCs are defective for lymphoid repopulating potential and show a myeloid lineage output bias. These cell autonomous abnormalities are exacerbated by physiologic aging and upon serial transplantation. The 5A3 deletion partially rescues defective repopulation in Gata2 mutant mice. 5A3+/del hematopoietic cells exhibit decreased expression of oxidative phosphorylation genes, increased levels of reactive oxygen species, and perturbed oxygen consumption. These studies provide the first functional data linking 7q22 deletions to MDS pathogenesis. DOI: http://dx.doi.org/10.7554/eLife.07839.001, eLife digest Stem cells in the bone marrow are essential for creating new blood cells. Myelodysplastic syndrome (MDS) is a common type of blood cancer in the elderly that occurs when blood cells fail to develop normally. Depending on which types of blood cells are affected, individuals with MDS may bleed more easily, feel weak and tired, or be unable to effectively fight off infections. Animals and plants store their genetic information in the form of chromosomes. Humans have 23 pairs of chromosomes, with one copy inherited from the mother, and the other from the father. The bone marrow cells of many people with MDS delete a section from one of their copies of chromosome 7. As this section contains many different genes, it is difficult to fully understand which specific genes contribute to the development of MDS when one copy is lost. Wong et al. have now genetically engineered mice to lack a section of one of their copies of chromosome 7 that is often missing in patients with MDS. Bone marrow cells from these mice exhibit many of the same abnormalities found in human MDS. For example, most of the immature blood stem cells expand, but these stem cells do not correctly specialize into mature blood cells—in particular, not enough immune cells are produced. The developing blood cells also have problems expressing several genes, including one that helps to protect the cells from damaging molecules called reactive oxygen species. These problems worsen as the mice age. These mice provide the first evidence directly linking the missing section of chromosome 7 to abnormalities found in MDS patients. Future studies using the mice will advance our understanding of how the loss of this section of chromosome 7 interacts with other genes involved in MDS to alter the course of this disease and how it responds to treatment. DOI: http://dx.doi.org/10.7554/eLife.07839.002

Fanconi Anemia (FA) is an autosomal recessive disorder characterized by cellular hypersensitivity to DNA cross-linking agents. Recent studies suggest that FA proteins share a common pathway with BRCA proteins. To study the in vivo role of the FA group A gene (Fanca), gene-targeting techniques were used to generate Fanca(tm1Hsc) mice in which Fanca exons 1-6 were replaced by a beta-galactosidase reporter construct. Fanca(tm1.1Hsc) mice were generated by Cre-mediated removal of the neomycin cassette in Fanca(tm1Hsc) mice. Fanca(tm1.1Hsc) homozygotes display FA-like phenotypes including growth retardation, microphthalmia and craniofacial malformations that are not found in other Fanca mouse models, and the genetic background affects manifestation of certain phenotypes. Both male and female mice homozygous for Fanca mutation exhibit hypogonadism, and homozygous females demonstrate premature reproductive senescence and an increased incidence of ovarian cysts. We showed that fertility defects in Fanca(tm1.1Hsc) homozygotes might be related to a diminished population of primordial germ cells (PGCs) during migration into the gonadal ridges. We also found a high level of Fanca expression in pachytene spermatocytes. Fanca(tm1Hsc) homozygous males exhibited an elevated frequency of mispaired meiotic chromosomes and increased apoptosis in germ cells, implicating a role for Fanca in meiotic recombination. However, the localization of Rad51, Brca1, Fancd2 and Mlh1 appeared normal on Fanca(tm1Hsc) homozygous meiotic chromosomes. Taken together, our results suggest that the FA pathway plays a role in the maintenance of reproductive germ cells and in meiotic recombination.

Despite the cloning of four disease-associated genes for Fanconi anemia (FA), the molecular pathogenesis of FA remains largely unknown. To study FA complementation group A using the mouse as a mode I system, we cloned and characterized the mouse homolog of the human FANCA cDNA, The mouse cDNA (Fanca) encodes a 161-kDa protein that shares 65% amino acid sequence identity with human FANCA. Fanca is located at the distal region of mouse chromosome 8 and has a ubiquitous pattern of expression in embryonic and adult tissues. Expression of the mouse DNA in human FA-A cells restores the cellular drug sensitivity to normal levels. Thus, the expression pattern, protein structure, chromosomal location, and function of FANCA are conserved in the mouse. We also isolated a novel zinc finger protein, Zfp276, which has five C2H2 domains. Interestingly, Zfp276 is situated in the Fanca locus, and the 3'UTR of its cDNA overlaps with the last four exons of Fanca in a tail-to-tail manner. Zfp276 is expressed in the same tissues as Fanca, but does not complement the mitomycin C (MMC)-sensitive phenotype of FA-A cells. The overlapping genomic organization between Zfp276 and Fanca may have relevance to the disease phenotype of FA. (C) 2000 Academic Press.

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer, with T lineage ALL (T-ALL) accounting for approximately 20% of cases. Many T-ALL patients present with high risk clinical features, and receive aggressive multi-agent genotoxic therapies that pose a substantial risk of long-term adverse health effects. Glucocorticoids (GCs) are an integral part of current treatment strategies for T-ALL and other lymphoid cancers. Despite decades of clinical use, the molecular mechanisms underlying GC efficacy and resistance are incompletely understood. Limited initial response to GCs correlates with poor outcome, and secondary GC resistance is frequently observed in relapsed patients. Thus, identifying drugs that either enhance initial responses or prevent resistance to GCs could significantly improve the treatment of T-ALL and other lymphoid malignancies. Our laboratory performed retroviral insertional mutagenesis (RIM) in wild-type (KrasWT) and KrasG12D "knock-in" mutant mice to generate a panel of primary transplantable T-ALLs that recapitulate the genetic heterogeneity found in human cancers. We used these reagents to assess the efficacy of treatment with the GC dexamethasone (DEX) alone and in combination with the PI3 kinase (PI3K) inhibitor GDC-0941. We observed a robust and significant overall response to 15 mg/kg/day DEX in cohorts of recipient mice transplanted with 10 independent parental KrasWT (n=5) and KrasG12D (n=5) T-ALLs, which was modestly enhanced by combined treatment with 125 mg/kg/day GDC-0941. Prolonged in vivo treatment resulted in outgrowth of 65 independent relapsed T-ALLs, many of which harbor novel retroviral integrations. We verified intrinsic drug resistance in a number of these relapsed leukemias by transplanting them into secondary recipients and treating these mice with 15 mg/kg/day DEX. Intriguingly, 23 (35%) of the T-ALLs that relapsed after an initial response to treatment exhibited markedly reduced GC receptor (GR) protein expression, suggesting a novel and common mechanism for evading GC-induced cell death. Similarly, analysis of T-ALL patient samples showed that low GR expression is rare in diagnostic specimens, but enriched in relapsed/refractory cases. We have identified two likely mechanisms of GR down-regulation in relapsed mouse T-ALLs including a nonsense mutation in the gene encoding GR and a retroviral integration in a putative distal GR promoter region that greatly reduces mRNA expression. We also performed transcriptome (RNA-seq) analysis in one sensitive parental and corresponding resistant KrasWT T-ALL after short-term in vivo DEX treatment, and observed a dramatic reduction in the number of differentially expressed genes in the resistant versus parental leukemia. We recently generated transcriptomes from this panel of 10 primary RIM-induced T-ALLs with the goal of identifying a GC response signature, and also to assess the ability of resistant cells that have lost or retained GR expression to activate this program. Finally, in contrast to previous studies in this genetically accurate in vivo preclinical model (Dail et al. Nature 2014), very few relapsed T-ALLs showed reduced Notch intracellular domain (NICD) expression relative to the corresponding parental leukemia suggesting that loss of Notch1 activation is not a major mechanism contributing to DEX resistance. Taken together, these data reveal an unexpected and common putative mechanism of GC resistance in T-ALL that can inform the development of treatment strategies for relapsed/refractory patients. We are currently focusing on analyzing transcriptome data and performing whole exome sequencing in order to uncover additional mechanisms of resistance to DEX, and then using these data to identify and investigate targeted inhibitors that might overcome GC resistance in vivo. Disclosures Dail: Genentech, Inc.: Employment. Sampath:Genentech: Employment.

Our molecular understanding of the how tumor suppressor gene (TSG) abnormalities contribute to myeloid malignancies is relatively limited. While the NF1 and TP53 TSGs follow the Knudson two-hit paradigm and undergo biallelic inactivation, there is increasing evidence that inactivation of a single allele of TSG such as RUNX1, PU.1 and RPS14 (haploinsufficiency) can also contribute to leukemogenesis. New technologies including high density single nucleotide polymorphism (SNP) arrays, RNA interference (RNAi) and chromosome engineering to develop mouse models with defined genetic rearrangements are emerging as potent tools for cloning and studying the function of TSGs. Notwithstanding these advances, the role of many chromosomal deletions that are commonly observed in myeloid malignancies remains uncertain, particularly the deletion of chromosomes 5, 7, 9 and 20. Since these deletions are often associated with resistance to current therapies, discovering the relevant TSGs and determining how they function in cell growth are high priorities.

MLL5 is a novel trithorax group gene and a candidate tumor suppressor gene located within a 2.5-Mb interval of chromosome band 7q22 that frequently is deleted in human myeloid malignancy. Here we show that inactivation of the Mll5 gene in mice results in a 30% reduction in the average representation of hematopoietic stem cells and in functional impairment of long-term hematopoietic repopulation potential under competitive conditions. Bone marrow cells from Mll5-deficient mice were defective in spleen colony-forming assays, and the mutant mice showed enhanced susceptibility to 5-fluorouracil–induced myelosuppression. Heterozygous and homozygous Mll5 mutant mice did not spontaneously develop hematologic cancers, and loss of Mll5 did not alter the phenotype of a fatal myeloproliferative disorder induced by oncogenic Kras in vivo. Collectively, the data reveal an important role for Mll5 in HSC homeostasis and provide a basis for further studies to explore its role in leukemogenesis.

Loss of heterozygosity (LOH) involving chromosome 16q23.4 occurs frequently in breast tumors, which suggests that this region may contain a tumor suppressor gene. Since ZFP276 is located in this region, we have therefore cloned and performed mutation analysis of its coding region in 70 breast tumors. One silent polymorphism and two alterations predicted to result in amino acid changes were detected. Absence of the wild-type allele in tumors carrying the E530D variant suggests a possible role for this change in tumorigenesis.

Fanconi anemia (FA) is a chromosomal instability syndrome characterized by the presence of pancytopenia, congenital malformations and cancer predisposition. Six genes associated with this disorder have been cloned, and mice with targeted disruptions of several of the FA genes have been generated. These mouse models display the characteristic FA feature of cellular hypersensitivity to DNA cross-linking agents. Although they do not develop hematological or developmental abnormalities spontaneously, they mimic FA patients in their reduced fertility. Studies using these animal models provide valuable insights into the involvement of apoptotic pathways in FA, and help characterize the defects in FA hematopoietic cells. In addition, mouse models are also useful for testing treatments for FA.

The myelodysplastic syndromes (MDS) are clonal hematologic neoplasms characterized by normal, increased or decreased bone marrow cellularity, ineffective production of one or more blood cell lineages, morphologic dysplasia, and a variable risk of progression to acute myeloid leukemia. Somatic monosomy 7 (-7) and deletions affecting the long arm of chromosome 7 [del(7q)] are highly prevalent cytogenetic abnormalities in de novo and therapy-related MDS that are associated with advanced age, prior exposure to alkylating agents, a high risk of leukemic transformation, and intrinsic drug resistance. Although recent genome-wide and targeted DNA sequencing efforts have uncovered mutations in genes involved in chromatin modification, transcription, RNA splicing, and signal transduction in MDS, extensive sequencing and expression analysis have not revealed recurring “second hit” mutations of any gene located on chromosome band 7q22, which is commonly deleted in patients with myeloid malignancies. Therefore, answering the long-standing question of how recurrent deletions of the long arm of chromosome 7 contribute to initiation, maintenance, and clinical outcome of MDS remains a fundamental challenge. Given the importance of understanding the biology of -7/del(7q) and the urgent need to develop new mechanism-based treatments for hematologic cancers with these deletions, we generated mice with a heterozygous germline deletion of a 2 Mb interval of chromosome band 5A3 syntenic to an interval of human chromosome band 7q22 commonly deleted in human patients (Blood 88:1930,1996). We find that 5A3 haploinsufficiency perturbs hematopoietic stem cell (HSC) development and function in vivo. 5A3+/del mice exhibit a reduced bone marrow cellularity, an expanded proportion of long-term HSCs, and an altered distribution of lineage-committed progenitors. 5A3+/del HSCs display impaired competitive reconstitution of lymphoid hematopoietic cells and a myeloid output bias, but also paradoxically show enhanced stem cell reconstitution upon aging and after serial transplantation. These defects are cell autonomous. Like WT HSCs, 5A3+/del HSCs exhaust their self-renewal potential by the tertiary round of transplantation. Taqman quantitative real-time PCR confirmed that all seven genes within the deleted interval that are expressed at detectable levels in HSC and multi-potent progenitors (MPP)(Mll5, Armc10, Psmc2, Dnajc2, Orc5l, Pmpcb and Napepld) showed ~50% reduction in transcripts in 5A3+/delHSC and MPP. Transcriptome sequencing (RNA-seq) analysis identified broad changes in lineage signature gene expression, as well as down-regulation of genes and pathways involved in oxidative phosphorylation (OXPHOS) in 5A3+/del HSCs. Whereas reactive oxygen species (ROS) are increased in aged 5A3+/del MPP, treatment of 5A3+/delmice with antioxidant N-Acetyl Cysteine failed to correct the HSC defects, suggesting that increased ROS is not the primary cause of these hematopoietic defects. Together, the abnormal properties of 5A3+/del hematopoietic cells support a mechanistic role of 7q22 deletions as contributing to cardinal features of MDS, which include impaired differentiation, myeloid lineage output bias, and a pronounced age-associated increase in disease incidence. Disclosures No relevant conflicts of interest to declare.

Abstract 2340 Monosomy 7 and deletion 7q [del(7q)] are among the most common cytogenetic alterations found in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). However, little is known regarding how chromosome 7 deletions contribute to the pathogenesis of myeloid malignancies. We harnessed chromosome engineering in mice to investigate the in vivo consequences of deleting a contiguous 2 Mb interval on chromosome 5A3 that is syntenic to a segment of chromosome band 7q22 that is commonly deleted in human myeloid malignancies (Blood 88:1930–5, 1996). This region contains 14 known genes, and homozygous deletion is embryonic lethal. Heterozygous 5A3+/del mice are grossly normal and have normal peripheral blood counts, but they have a 20% reduction in spleen weight and a 25% reduction in bone marrow (BM) cellularity per femur. We did not observe a significant difference in the frequency of phenotypic long-term hematopoietic stem cells (LT-HSC) (c-kit+, Sca-1+, Lin−, CD150+, CD41−, CD48−) between 5A3+/del mice and their wild-type (WT) littermates; however, there is a ∼30% reduction in the frequency of multipotent progenitors (MPP) (c-kit+, Sca-1+, Lin−, CD150−, CD41−, CD48−), suggesting a defect in the transition from LT-HSC to MPP. Since 5A3+/del mice did not spontaneously develop evidence of hematologic malignancy in >2 years of observation, and transplantation enforces enhanced HSC cycling which may reveal subtle HSC phenotypes not obvious in the steady state, we performed competitive repopulation assays to directly assess the function of 5A3+/del HSC in vivo. Unfractionated test BM from WT or 5A3+/del animals (CD45.2) were mixed with WT competitor BM (CD45.1) at a 1:1 or 1:2 ratio, followed by transplantation into lethally irradiated WT recipients (CD45.1+2). Although 5A3+/del BM supported reconstitution of all hematopoietic lineages 6 months after adoptive transfer, reductions of 46% (p=0.0003) and 45% (p=0.0010) in comparison to WT were observed in the repopulation of B and T cell lineages, respectively. Reconstitution of the myeloid lineage was reduced to a lesser extent (25% reduction; p=0.0921), and analysis of the lineage distribution of myeloid, B, and T cell lineages within 5A3+/del derived cells revealed a myeloid lineage skewing reminiscent of the pattern of repopulation exhibited by aged HSC. Interestingly, a progressive decline in the repopulating ability of 5A3+/del BM upon cell differentiation was observed: when 5A3+/del test BM were transplanted in a more stringent 1:2 test:competitor ratio, we observed a 29% reduction (p=0.1657) in contribution to the K+L−S+ (c-kit+, Lin−, Sca-1+) stem cell compartment in comparison to WT, but a 39% reduction (p=0.0498) in the more mature progenitor K+L−S− (c-kit+, Lin-, Sca-1−) population. To investigate if the marked deficit in repopulation is intrinsic to the LT-HSC population and to assess if haploinsufficiency of the 5A3 region in the microenvironment modulates the phenotype, we purified and transplanted 15 WT or 5A3+/del LT-HSC together with 250,000 unfractionated WT BM competitors into WT or 5A3+/del recipients respectively. Transplantation of 5A3+/del LT-HSC demonstrated a similar trend of global repopulating deficit and overall pattern of altered lineage distribution regardless of the genotype of the recipient, which infers a dominant cell intrinsic mechanism of action of the 5A3 deletion. Meanwhile, transplantation of WT LT-HSC into 5A3+/del recipients did not significantly modulate repopulating potential. To determine if 5A3+/del LT-HSC is defective in self-renewal, we analyzed reconstitution in the LT-HSC compartment. 5A3+/del LT-HSC repopulated the LT-HSC compartment as efficiently as WT controls regardless of the recipient genotype, and subfractionation of the 5A3+/del derived KLS population to LT-HSC and MPP subsets revealed a significantly biased distribution towards LT-HSC by 1.39-fold (p=0.0146), suggesting increased self-renewal. Together, these data demonstrate that haploinsufficiency of the 5A3 interval leads to defective HSC function including a perturbed stem cell compartment, diminished overall repopulating potential and a myeloid-biased differentiation pattern. The phenotypic resemblance to aged HSC is intriguing given the increased incidence of MDS with monosomy 7 in elderly individuals. Disclosures: No relevant conflicts of interest to declare.

Identifying tumor suppressor genes from intervals that are deleted in human hematologic malignancies such as 5q, 7q, 9q, and 20q has proven extremely challenging, and several laboratories have implicated haploinsufficiency as a likely mechanism. Chromosome engineering, which involves performing sequential rounds of gene targeting to insert loxP sites at flanking loci in mouse embryonic stem (ES) cells and using Cre recombination to delete the intervening sequences, was recently used to successfully interrogate the 1p36 interval in human solid tumors (Cell128(3):459–75, 2007). Monosomy 7 and deletion 7q [del(7q)] are among the most common cytogenetic alterations found in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Cytogenetic analysis of patients who developed myeloid disorders with del(7q) uncovered a 2.5 Mb commonly deleted segment (CDS) within 7q22 (Blood88(6):1930–5, 1996), suggesting that this region plays an important role in leukemogenesis. To investigate the in vivo consequences of somatic loss of this interval, we generated a 5A3flox mouse model that harbor loxP sites flanking a ∼2 Mb interval on mouse chromosome 5A3 that is syntenic to the human 7q22 CDS. We intercrossed these mice with the interferon inducible Mx1-Cre transgenic strain, and injected these mice with polyinosinic-polycytidylic acid (pIpC) to delete the region in the hematopoietic compartment. The desired recombination is relatively inefficient; however, hematopoietic cells with loss of this region persist in the stem/progenitor compartment for over 1 year and are transplantable. We neither observed a block in differentiation nor clonal outgrowth of mutant hematopoietic cells, suggesting that additional mutations are necessary to initiate leukemia. We initiated AML in these mice by introducing additional genetic lesions using retroviral insertional mutagenesis, and we are characterizing these leukemias to study the effects of the 5A3 deletion on leukemogenesis and to clone cooperating genes. Chromosome engineering is a robust strategy for modeling leukemia-associated deletions in vivo, and for interrogating how loss of a specific interval alters hematopoietic growth. We are using the 5A3 strain to analyze candidate myeloid tumor suppressor genes from chromosome 7q and to uncover genes and pathways that cooperate in leukemogenesis.