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The most important prognostic factor for atherosclerotic cardiovascular disease is age, independent of all other recognized risk factors. Recently, exome sequence analyses showed that somatic mutations in blood cells, a process termed clonal hematopoiesis, are common and increase in prevalence with age, with at least 1 in 10 adults older than 70 years affected. Carriers of clonal hematopoiesis have been shown to be not only at heightened risk for hematologic malignancy but also at increased risk for atherosclerotic cardiovascular disease. Here, we review the prior literature of clonal selection and expansion of hematopoietic stem cells and the evidence supporting its causal association with atherosclerotic cardiovascular disease.

The pattern of somatic mutations observed at diagnosis of acute myeloid leukemia (AML) has been well-characterized. However, the premalignant mutational landscape of AML and its impact on risk and time to diagnosis is unknown. Here we identified 212 women from the Women’s Health Initiative who were healthy at study baseline, but eventually developed AML during follow-up (median time: 9.6 years). Deep sequencing was performed on peripheral blood DNA of these cases and compared to age-matched controls that did not develop AML. We discovered that mutations in IDH1, IDH2, TP53, DNMT3A, TET2 and spliceosome genes significantly increased the odds of developing AML. All subjects with TP53 mutations (n = 21 out of 21 patients) and IDH1 and IDH2 (n = 15 out of 15 patients) mutations eventually developed AML in our study. The presence of detectable mutations years before diagnosis suggests that there is a period of latency that precedes AML during which early detection, monitoring and interventional studies should be considered.

Activating mutations in genes encoding G protein α (Gα) subunits occur in 4-5% of all human cancers, but oncogenic alterations in Gβ subunits have not been defined. Here we demonstrate that recurrent mutations in the Gβ proteins GNB1 and GNB2 confer cytokine-independent growth and activate canonical G protein signaling. Multiple mutations in GNB1 affect the protein interface that binds Gα subunits as well as downstream effectors and disrupt Gα interactions with the Gβγ dimer. Different mutations in Gβ proteins clustered partly on the basis of lineage; for example, all 11 GNB1 K57 mutations were in myeloid neoplasms, and seven of eight GNB1 I80 mutations were in B cell neoplasms. Expression of patient-derived GNB1 variants in Cdkn2a-deficient mouse bone marrow followed by transplantation resulted in either myeloid or B cell malignancies. In vivo treatment with the dual PI3K-mTOR inhibitor BEZ235 suppressed GNB1-induced signaling and markedly increased survival. In several human tumors, mutations in the gene encoding GNB1 co-occurred with oncogenic kinase alterations, including the BCR-ABL fusion protein, the V617F substitution in JAK2 and the V600K substitution in BRAF. Coexpression of patient-derived GNB1 variants with these mutant kinases resulted in inhibitor resistance in each context. Thus, GNB1 and GNB2 alterations confer transformed and resistance phenotypes across a range of human tumors and may be targetable with inhibitors of G protein signaling.

BACKGROUND: Clonal hematopoiesis of indeterminate potential (CHIP), defined by the presence of an expanded somatic blood cell clone in those without other hematologic abnormalities, is common in older individuals and associates with an increased risk of developing hematologic cancer. We previously found preliminary evidence for an association of CHIP with human atherosclerotic cardiovascular disease, but the nature of this association was unclear. METHODS: We used whole exome sequencing to detect the presence of CHIP in peripheral blood cells and associated this with coronary heart disease in four case-control studies together comprising 4,794 cases and 3,537 controls. To assess causality, we perturbed the function of Tet2, the second most commonly mutated gene linked to clonal hematopoiesis, in the hematopoietic cells of atherosclerosis-prone mice. RESULTS: In nested case-control analyses from two prospective cohorts, carriers of CHIP had a 1.9-fold (95% confidence interval 1.4–2.7) increased risk of coronary heart disease compared to non-carriers. In two retrospective case-control cohorts for early-onset myocardial infarction, those with CHIP had a 4.0-fold greater risk (95% confidence interval 2.4–6.7) of having myocardial infarction. Mutations in DNMT3A, TET2, ASXL1, and JAK2 were each individually associated with coronary heart disease. Those with clonal hematopoiesis also had increased coronary artery calcification, a marker of coronary atherosclerosis burden. Hyperlipidemic mice engrafted with Tet2−/− or Tet2+/− bone marrow developed larger atherosclerotic lesions in the aortic root and aorta than mice receiving control marrow. Analyses of Tet2−/− macrophages demonstrated elevated expression of several chemokine and cytokine genes that contribute to atherosclerosis. CONCLUSIONS: Clonal hematopoiesis robustly associates with coronary heart disease in humans and causes accelerated atherosclerosis in mice.

Purpose Clonal hematopoiesis of indeterminate potential (CHIP) is an age-related condition characterized by somatic mutations in the blood of otherwise healthy adults. We hypothesized that in patients undergoing autologous stem-cell transplantation (ASCT) for lymphoma, CHIP at the time of ASCT would be associated with an increased risk of myelodysplastic syndrome and acute myeloid leukemia, collectively termed therapy-related myeloid neoplasm (TMN), and other adverse outcomes. Methods We performed whole-exome sequencing on pre- and post-ASCT samples from 12 patients who developed TMN after autologous transplantation for Hodgkin lymphoma or non-Hodgkin lymphoma and targeted sequencing on cryopreserved aliquots of autologous stem-cell products from 401 patients who underwent ASCT for non-Hodgkin lymphoma between 2003 and 2010. We assessed the effect of CHIP at the time of ASCT on subsequent outcomes, including TMN, cause-specific mortality, and overall survival. Results For six of 12 patients in the exome sequencing cohort, mutations found in the TMN specimen were also detectable in the pre-ASCT specimen. In the targeted sequencing cohort, 120 patients (29.9%) had CHIP at the time of ASCT, which was associated with an increased rate of TMN (10-year cumulative incidence, 14.1% v 4.3% for those with and without CHIP, respectively; P = .002). Patients with CHIP had significantly inferior overall survival compared with those without CHIP (10-year overall survival, 30.4% v 60.9%, respectively; P < .001), including increased risk of death from TMN and cardiovascular disease. Conclusion In patients undergoing ASCT for lymphoma, CHIP at the time of transplantation is associated with inferior survival and increased risk of TMN.

Importance Clonal hematopoiesis (CH) has been recently described as a novel driver for cancer and cardiovascular disease (CVD). Clonal hematopoiesis is a common, age-associated disorder marked by expansion of hematopoietic clones carrying recurrent somatic mutations. Current literature suggests that patients with CH have a higher risk of subsequent hematological malignant conditions and mortality attributable to excess CVD. This review discusses the association of cancer with CVD with CH as a potential unifying factor. Observations The prevalence of CH varies based on the sequencing depth, diagnostic criteria, and patient age and ranges from less than 1% in those younger than 40 years to more than 15% to 20% in those 90 years and older. Clonal hematopoiesis is associated with a 0.5% to 1.0% absolute annual risk of hematological malignant condition and a 2-fold to 4-fold higher risk of coronary artery disease, stroke, and CVD deaths, independent of traditional cardiovascular risk factors. In fact, CH appears to have a relative risk similar to that of traditional cardiovascular risk factors for CVD. Experimental studies suggest that the link between CVD and CH is causal, with inflammation as 1 potential mechanism. There may be also a link between CH and CVD in survivors of cancer; however, data to support this association are currently limited. Conclusions and Relevance Clonal hematopoiesis represents a premalignant state, with carriers having an increased risk of hematological malignant conditions. Although most carriers will not develop a malignant condition, CH confers an increased risk of CVD, possibly via inflammation. Clonal hematopoiesis may also contribute to CVD in survivors of cancer, although this hypothesis requires validation. Clinically, as advanced sequencing techniques become available, CH may pave the way for precision medicine in the field of cardio-oncology.

CD47 exerts different effects on disease in distinct cell types and locations and during different stages of experimental autoimmune encephalomyelitis., Comparison of transcriptomic and proteomic data from pathologically similar multiple sclerosis (MS) lesions reveals down-regulation of CD47 at the messenger RNA level and low abundance at the protein level. Immunohistochemical studies demonstrate that CD47 is expressed in normal myelin and in foamy macrophages and reactive astrocytes within active MS lesions. We demonstrate that CD47−/− mice are refractory to experimental autoimmune encephalomyelitis (EAE), primarily as the result of failure of immune cell activation after immunization with myelin antigen. In contrast, blocking with a monoclonal antibody against CD47 in mice at the peak of paralysis worsens EAE severity and enhances immune activation in the peripheral immune system. In vitro assays demonstrate that blocking CD47 also promotes phagocytosis of myelin and that this effect is dependent on signal regulatory protein α (SIRP-α). Immune regulation and phagocytosis are mechanisms for CD47 signaling in autoimmune neuroinflammation. Depending on the cell type, location, and disease stage, CD47 has Janus-like roles, with opposing effects on EAE pathogenesis.

CD47, a “don't eat me” signal for phagocytic cells, is expressed on the surface of all human solid tumor cells. Analysis of patient tumor and matched adjacent normal (nontumor) tissue revealed that CD47 is overexpressed on cancer cells. CD47 mRNA expression levels correlated with a decreased probability of survival for multiple types of cancer. CD47 is a ligand for SIRPα, a protein expressed on macrophages and dendritic cells. In vitro, blockade of CD47 signaling using targeted monoclonal antibodies enabled macrophage phagocytosis of tumor cells that were otherwise protected. Administration of anti-CD47 antibodies inhibited tumor growth in orthotopic immunodeficient mouse xenotransplantation models established with patient tumor cells and increased the survival of the mice over time. Anti-CD47 antibody therapy initiated on larger tumors inhibited tumor growth and prevented or treated metastasis, but initiation of the therapy on smaller tumors was potentially curative. The safety and efficacy of targeting CD47 was further tested and validated in immune competent hosts using an orthotopic mouse breast cancer model. These results suggest all human solid tumor cells require CD47 expression to suppress phagocytic innate immune surveillance and elimination. These data, taken together with similar findings with other human neoplasms, show that CD47 is a commonly expressed molecule on all cancers, its function to block phagocytosis is known, and blockade of its function leads to tumor cell phagocytosis and elimination. CD47 is therefore a validated target for cancer therapies.

Clonal hematopoiesis of indeterminate potential (CHIP) was recently identified as a major risk factor for development of both hematologic malignancies and atherosclerotic cardiovascular disease in humans. The most commonly mutated gene in CHIP, DNMT3A, is a de novo DNA methyltransferase. The second most commonly mutated gene is TET2, an enzyme which can lead to loss of DNA methylation, and thus is thought to have an opposing biochemical function to DNMT3A. Surprisingly, mutations in both genes lead to convergent phenotypes, such as clonal expansion of mutated stem cells, increased risk of malignant transformation, and increased risk of coronary heart disease. A molecular mechanism linking CHIP and cardiovascular disease has been explored only for loss of function mutations in the Tet2 gene (Jaiswal et al., NEJM 2017; Fuster et al., Science 2017). Here we tested the ability of null mutations in Dnmt3a to contribute to atherosclerosis in hypercholesteremic mice. We further explored the biological basis for this association through gene expression analyses and single-cell RNA sequencing. To model cardiovascular disease associated with DNMT3A-mutated CHIP, atherosclerosis-prone Ldlr-/- mice received bone marrow from Dnmt3a+/+ mice (WT), or from Dnmt3a-/- mice (KO) and WT mice in a 1:9 ratio to mimic a typical variant allele fraction observed in human CHIP. Mice then consumed a high-fat, high-cholesterol diet (HFD), and underwent assessment of atherosclerosis. At 9 weeks, mice that had received 10% Dnmt3a-/- bone marrow displayed an average lesion size that was 40% larger compared to mice receiving control marrow only (p=0.04). The increase in lesion size resembles that we previously observed in mice receiving Tet2-/- marrow (Jaiswal et al., NEJM 2017). De novo DNA methylation by Dnmt3a can alter gene expression. To elucidate how such changes may accelerate atherosclerosis, we first performed transcriptome analysis using bulk RNA sequencing of cholesterol-stimulated bone marrow derived macrophages (BMDM) from either WT or KO mice. BMDMs lacking Dnmt3a showed significantly augmented expression of genes belonging to the CXC chemokine cluster, Cxcl1, Cxcl2 and Cxcl3, as well as increases in mRNAs encoding canonical pro-inflammatory cytokines Il1b and Il6. These changes mirrored those we saw in macrophages lacking Tet2 (Jaiswal et al., NEJM 2017). We next asked how transcriptomic changes observed using the ex vivo BMDM system translated into the in vivo lesional environment. Single-cell RNA sequencing (10X Genomics) was performed on atherosclerotic aortae from mice that had been competitively transplanted with WT, Dnmt3a-/-, or Tet2-/- marrow at a 1:9 ratio. Clustering demonstrated broad changes in lesional immune cell composition in mice harboring CHIP. Lack of either Tet2 or Dnmt3a substantially expanded the myeloid compartment, containing cells that drive atherogenesis. A reciprocal reduction mainly affecting T lymphocyte populations accompanied this expansion. Within the myeloid cell compartment, Dnmt3a-/- or Tet2-/- donor cells, but not WT donor cells, gave rise to a distinct lesional macrophage population. These cells expressed markers associated with tissue-resident macrophages (Mrc1, Lyve1, F13a1), but also highly expressed several inflammatory mediators (Cxcl1, Pf4, Ccl2, Ccl7, Ccl8), and a characteristic set of transcription factors (Jun, Fos, Egr1). Overall, the present study reveals broad changes to the lesional cellular composition and transcriptome induced by the most common CHIP mutations, and provides novel insight into the mechanisms by which CHIP accelerates atherosclerosis. Despite exerting opposite catalytic functions, lack of Dnmt3a or of Tet2 function lead to a myriad of similar downstream transcriptomic and cellular changes. These results indicate that mutations in Dnmt3a and Tet2 accelerate atherosclerosis through convergent mechanisms. Disclosures No relevant conflicts of interest to declare.

Tumor immunosurveillance is a well-established mechanism for regulation of tumor growth. In this regard, most studies have focused on the role of T- and NK-cells as the critical immune effector cells. However, macrophages play a major role in the recognition and clearance of foreign, aged, and damaged cells. Macrophage phagocytosis is negatively regulated via the receptor SIRPalpha upon binding to CD47, a ubiquitously expressed protein. We recently showed that CD47 is up-regulated in myeloid leukemia and migrating hematopoietic progenitors, and that the level of protein expression correlates with the ability to evade phagocytosis. These results implicate macrophages in the immunosurveillance of hematopoietic cells and leukemias. The ability of macrophages to phagocytose tumor cells might be exploited therapeutically by blocking the CD47-SIRPalpha interaction.

Recent genetic analyses of large populations have revealed that somatic mutations in hematopoietic cells leading to clonal expansion are commonly acquired during human aging. Clonally restricted hematopoiesis is associated with an increased risk of subsequent diagnosis of myeloid or lymphoid neoplasia and increased all-cause mortality. Although myelodysplastic syndromes (MDS) are defined by cytopenias, dysplastic morphology of blood and marrow cells, and clonal hematopoiesis, most individuals who acquire clonal hematopoiesis during aging will never develop MDS. Therefore, acquisition of somatic mutations that drive clonal expansion in the absence of cytopenias and dysplastic hematopoiesis can be considered clonal hematopoiesis of indeterminate potential (CHIP), analogous to monoclonal gammopathy of undetermined significance and monoclonal B-cell lymphocytosis, which are precursor states for hematologic neoplasms but are usually benign and do not progress. Because mutations are frequently observed in healthy older persons, detection of an MDS-associated somatic mutation in a cytopenic patient without other evidence of MDS may cause diagnostic uncertainty. Here we discuss the nature and prevalence of CHIP, distinction of this state from MDS, and current areas of uncertainty regarding diagnostic criteria for myeloid malignancies.

Myelodysplastic syndrome (MDS) has long been presumed to be a stem cell disorder, but rigorous formal proof has been lacking. In this issue of Cancer Cell, Woll and colleagues demonstrate that driver mutations occurring in MDS definitively occur in cells with a stem cell phenotype.

BACKGROUND: The incidence of hematologic cancers increases with age. These cancers are associated with recurrent somatic mutations in specific genes. We hypothesized that such mutations would be detectable in the blood of some persons who are not known to have hematologic disorders. METHODS: We analyzed whole-exome sequencing data from DNA in the peripheral-blood cells of 17,182 persons who were unselected for hematologic phenotypes. We looked for somatic mutations by identifying previously characterized single-nucleotide variants and small insertions or deletions in 160 genes that are recurrently mutated in hematologic cancers. The presence of mutations was analyzed for an association with hematologic phenotypes, survival, and cardiovascular events. RESULTS: Detectable somatic mutations were rare in persons younger than 40 years of age but rose appreciably in frequency with age. Among persons 70 to 79 years of age, 80 to 89 years of age, and 90 to 108 years of age, these clonal mutations were observed in 9.5% (219 of 2300 persons), 11.7% (37 of 317), and 18.4% (19 of 103), respectively. The majority of the variants occurred in three genes: DNMT3A, TET2, and ASXL1. The presence of a somatic mutation was associated with an increase in the risk of hematologic cancer (hazard ratio, 11.1; 95% confidence interval [CI], 3.9 to 32.6), an increase in all-cause mortality (hazard ratio, 1.4; 95% CI, 1.1 to 1.8), and increases in the risks of incident coronary heart disease (hazard ratio, 2.0; 95% CI, 1.2 to 3.4) and ischemic stroke (hazard ratio, 2.6; 95% CI, 1.4 to 4.8). CONCLUSIONS: Age-related clonal hematopoiesis is a common condition that is associated with increases in the risk of hematologic cancer and in all-cause mortality, with the latter possibly due to an increased risk of cardiovascular disease. (Funded by the National Institutes of Health and others.).

Background Clonal hematopoiesis of indeterminate potential (CHIP) is an age-related phenomenon characterized by the presence of somatic mutations in peripheral blood (PMIDs: 25426837, 25426838). Although CHIP was originally defined in healthy older adults without cytopenias, it can be found in other contexts as well. For example, one recent report described four patients with therapy-related myeloid neoplasm (TMN) arising after treatment for other cancers, all of which were driven by TP53 mutations that could be found at very low levels in samples drawn years before the development of TMN (PMID: 25487151). However, there has not yet been a more systematic study of CHIP in this type of context. In this study, we sought to understand how CHIP behaves and influences outcomes in the context of autologous stem cell transplantation (ASCT), arguably the most extreme selective pressure that can be studied in the context of native hematopoiesis. We hypothesized that in patients with Non-Hodgkin Lymphoma (NHL) undergoing ASCT, the presence of CHIP at the time of transplantation would be associated with an increased risk of TMN and other adverse outcomes. Methods We analyzed exome sequencing data from 10 patients with TMN after ASCT (City of Hope Cancer Center, Duarte, CA), and performed targeted sequencing of 116 genes on banked, mobilized peripheral blood from an additional 401 patients with NHL who underwent ASCT (Dana Farber Cancer Institute, Boston, MA), to determine whether there is a clonal connection between CHIP at the time of ASCT and subsequent TMN, and to determine whether the presence of CHIP at the time of ASCT influences subsequent outcomes. Results In 7 of 10 TMN patients for whom we analyzed exome sequencing data, mutations present at the time of TMN were also detectable in the pre-ASCT sample. PPM1D, a key mediator of the DNA damage pathway, was mutated in 2 patients, as was TP53 (2 patients), TET2 (2 patients) and PRPF8 (1 patient). In our larger cohort of 401 unselected ASCT patients, CHIP was common (121 patients, 30.2%) and was associated with older age but not with other demographic or treatment-related factors. PPM1D was the most commonly mutated gene (54 mutations in 48 patients). In the ASCT cohort of 401 patients, 18 patients developed TMN. The presence of CHIP at the time of ASCT significantly increased this risk: the 10-year cumulative incidence of TMN, with death and allogeneic transplant as competing risks, was 12.4% for patients with CHIP, compared to 3.5% for patients without CHIP (P=0.002, Figure 1A). Moreover, the presence of CHIP at the time of ASCT conferred significant risks beyond TMN alone, as patients with CHIP had significantly inferior overall survival compared to patients without CHIP (10-year OS 30.6% versus 60.9%, P=0.0003, Figure 1B). This difference was driven primarily by late mortality and not by an increased risk of relapse or by the difference in rate of TMN. Although other variables were associated with OS in univariate analysis, multivariate analysis in a Cox proportional hazards model showed that only older age (60 or above), aggressive lymphoma, and presence of CHIP were significantly associated with survival. Conclusion We show that CHIP at the time of ASCT for NHL is common and is associated with an increased risk of TMN and decreased overall survival independent of the TMN risk. These results have substantial clinical and translational implications. They suggest the need to specifically study the connection between CHIP and lymphoma more deeply, which could be accomplished by assessing CHIP in patients with newly diagnosed lymphoma prior to the administration of any chemotherapy or mobilizing agents. They also suggest the need to consider alternative therapeutic approaches for patients with lymphoma and a high risk of TMN who are being considered for ASCT. Finally, they underscore the need to study clonal hematopoiesis in the context of treatment for other cancers to determine whether these results may be relevant to an even larger number of patients. Disclosures Lindsley: MedImmune: Research Funding; Takeda Pharmaceuticals: Consultancy. Mar:H3 Biomedicine: Other: Spouse's employment. LaCasce:Forty Seven: Consultancy; Seattle Genetics: Consultancy; Seattle Genetics: Consultancy. Koreth:LLS: Research Funding; amgen inc: Consultancy; takeda pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; kadmon corp: Membership on an entity's Board of Directors or advisory committees; prometheus labs inc: Research Funding; millennium pharmaceuticals: Research Funding. Ritz:Kiadis: Membership on an entity's Board of Directors or advisory committees. Soiffer:Kiadis: Membership on an entity's Board of Directors or advisory committees; Juno: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

One of the adverse consequences of chemotherapy exposure is the development of therapy-related myeloid neoplasms (t-MNs). However, the cause and origin of most t-MNs are unknown, and the prognosis remains dismal. Novel work has shown that in addition to TP53, PPM1D is selectively mutated in 15% of therapy related MDS (Lindsley et al., ASH Abstract). Truncating mutations of PPM1D are also found to commonly occur in clonal hematopoiesis (Jaiswal et al., NEJM 2014; Genovese et al., NEJM 2014; Xie et al., Nat Med 2014), as well as in the blood of cancer patients, particularly after exposure to chemotherapy (Ruark et al., Nature 2013; Swisher et al., JAMA Oncol 2016). We hypothesized that PPM1D truncating mutations confer chemotherapy resistance, causing selective outgrowth of PPM1D mutant hematopoietic stem cells in states of genotoxic stress. In addition, since PPM1D mutations lead to a gain of function, we examined the potential of targeting PPM1D-mutant cells pharmacologically. The protein phosphatase PPM1Dis a direct regulator of TP53 activity and the DNA damage response pathway (Fiscella et al., PNAS 1997). Consequently, gain-of-function PPM1D mutations lead to decreased TP53 activity. To examine whether PPM1D mutations drive chemotherapy resistance through an abrogation of the TP53 dependent DNA damage response, we engineered PPM1D-mutant subclones using the CRISPR-Cas9 system in the TP53 wild-type AML cell line MOLM-13. PPM1D exon 6 truncation led to increased expression of PPM1D and resistance to DNA damaging agents, including cytarabine, cyclophosphamide and cisplatin. In addition, PPM1D-mutant cells exhibited a selective advantage over wild-type cells in the presence of chemotherapy, expanding 100-fold over a 24-day period (Fig. 1). While treatment with chemotherapy induced phosphorylation of Chk1 and p53, cell cycle arrest and apoptosis in wild-type cells, this response was abrogated in PPM1D-mutant cells due to gain of function of PPM1D. Using phosphoproteomic analysis, we further demonstrate decreased phosphorylation of known and novel targets in PPM1D-mutant compared to wild-type cells. We next investigated the effect of PPM1D mutation on normal marrow progenitors in response to chemotherapy treatment in vivo. We performed a competition experiment in which mouse bone marrow c-Kit+ cells expressing Cas9 were transduced with guide RNAs targeting exon 6 of PPM1D or a control guide, and then transplanted into mice in a 1:5 ratio. We observed a selective outgrowth of PPM1D-mutant myeloid cells in the peripheral blood of mice after exposure to chemotherapy. In addition, we found expansion of PPM1D-mutant cells in the lineage negative, c-Kit+ Sca-1+ population, which is enriched for hematopoietic stem cells and multipotent progenitors, indicating that PPM1D-mutant stem and progenitor cells have a competitive advantage over wild-type cells after exposure to genotoxic stress. The generation of a selective, allosteric inhibitor of PPM1D (Gilmartin et al., Nat Chem Biol 2014) allowed us to examine whether pharmacologic inhibition of PPM1D decreases the chemotherapy resistance or survival of PPM1D-mutant cells. We found that PPM1D-mutant cells have a significantly increased sensitivity to PPM1D inhibition when compared to wild-type controls. In addition, PPM1D inhibitor treatment was able to re-sensitize mutant cells to chemotherapy and abrogate the selective outgrowth of PPM1D-mutant cells during chemotherapy exposure. Lastly, we demonstrate that the proteome-wide phosphorylation profile characteristic of PPM1D-mutant cells can be reversed through treatment with the PPM1D inhibitor. In sum, these results demonstrate that PPM1D mutations confer a competitive advantage to hematopoietic stem cells undergoing genotoxic stress by abrogating the DNA damage response, and are likely to be the initiating mutation in a large proportion of t-MNs. Due to the gain-of-function nature of this mutation, PPM1D-mutant cells are differentially sensitive to treatment with a PPM1D inhibitor. PPM1D inhibition may therefore provide an opportunity for the prevention and targeted treatment of hematologic malignancies that harbor PPM1D mutations. Figure 1 PPM1D mutant cells have a competitive advantage under the selective pressure of chemotherapy (cytarabine) treatment. Figure 1. PPM1D mutant cells have a competitive advantage under the selective pressure of chemotherapy (cytarabine) treatment. Disclosures No relevant conflicts of interest to declare.

Under normal physiologic conditions, cellular homeostasis is partly regulated by balancing pro- and anti-phagocytic signals. CD47 is highly expressed on several human cancers including acute myeloid leukemia, non-Hodgkin lymphoma, and bladder cancer, allowing cancer cells to evade phagocytosis by the innate immune system. Blockade of CD47 with a monoclonal antibody enables phagocytosis of cancer cells and leads to in vivo tumor elimination, but leaves most normal cells unaffected. In order for target cells to be phagocytosed upon blockade of an anti-phagocytic signal, we postulate that the cells must also display a potent pro-phagocytic signal. Here we identify calreticulin as a pro-phagocytic signal highly expressed on the surface of several human cancers including acute myeloid and lymphoblastic leukemias, chronic myeloid leukemia, non-Hodgkin lymphoma (NHL), bladder cancer, glioblastoma, and ovarian cancer, but minimally expressed on most normal cells. Increased CD47 expression correlated with high calreticulin levels on cancer cells, and was necessary for protection from calreticulin-mediated phagocytosis. Phagocytosis induced by anti-CD47 antibody required the interaction of target cell calreticulin with its receptor low density lipoprotein-receptor related protein (LRP) on phagocytic cells, as blockade of the calreticulin/LRP interaction prevented anti-CD47 antibody mediated phagocytosis. Lastly, increased calreticulin expression was an adverse prognostic factor in diverse tumors including neuroblastoma, bladder cancer, and NHL. These findings identify calreticulin as the dominant pro-phagocytic signal on several human cancers, provide an explanation for the selective targeting of tumor cells by anti-CD47 antibody, and highlight the balance between pro- and anti-phagocytic signals in the immune evasion of cancer.

SummaryMacrophages clear pathogens and damaged or aged cells from the blood stream via phagocytosis. Cell-surface CD47 interacts with its receptor on macrophages, SIRPα, to inhibit phagocytosis of normal, healthy cells. We find that mobilizing cytokines and inflammatory stimuli cause CD47 to be transiently upregulated on mouse hematopoietic stem cells (HSCs) and progenitors just prior to and during their migratory phase, and that the level of CD47 on these cells determines the probability that they are engulfed in vivo. CD47 is also constitutively upregulated on mouse and human myeloid leukemias, and overexpression of CD47 on a myeloid leukemia line increases its pathogenicity by allowing it to evade phagocytosis. We conclude that CD47 upregulation is an important mechanism that provides protection to normal HSCs during inflammation-mediated mobilization, and that leukemic progenitors co-opt this ability in order to evade macrophage killing.

Hematological malignancies are associated with recurrent somatic mutations in specific genes, and may be preceded by a pre-malignant state in which only the initial driver mutation(s) are present. For example, monoclonal gammopathy of unknown significance often precedes multiple myeloma and monoclonal B-lymphocytosis can precede chronic lymphocytic leukemia. Recent sequencing studies have identified genes that are recurrently mutated in acute myeloid leukemia, myelodysplastic syndromes, myeloproliferative neoplasms, acute lymphoblastic leukemia, and other hematological neoplasms. We hypothesized that a pre-malignant state comprised of a clonal expansion of cells harboring some of these recurrent mutations would be detectable in the blood of elderly individuals not known to have hematological disorders. To address this question, we analyzed whole exome sequencing data from peripheral blood cell DNA of 17,182 individuals. Most of these were sequenced for type 2 diabetes genetic association studies and were therefore unselected for hematological phenotypes. We looked for candidate somatic variants by identifying previously characterized single nucleotide variants (SNVs) and small insertions/deletions (indels) in 160 genes recurrently mutated in hematological malignancies. The presence of these variants was analyzed for association to hematological phenotypes, survival, and cardiovascular events. We identified a total of 805 candidate somatic variants (hereafter referred to as mutations) from 746 individuals in 73 genes. Somatic mutations were rarely detected in individuals younger than 40, but rose appreciably with age (Figure 1). At ages 70-79, 80-89, and 90-108 these clonal mutations were observed in 9.6% (220 out of 2299), 11.7% (37 out of 317), and 18.4% (19 out of 103) of individuals, respectively. The majority of the variants occurred in 3 genes: DNMT3A (403 variants), TET2 (72 variants), and ASXL1 (62 variants). The median variant allele fraction for the detected somatic mutations was 0.09, from which we infer that the pathologic clone represents on average 18% of circulating white blood cells. Clinical outcome data was available on a subset of subjects, with a median follow-up period of 8 years. Carrying a somatic mutation was associated with increased risk of developing a hematological malignancy (hazard ratio [HR] 11, 95% confidence interval [95% CI] 3.9-33 by competing risks regression). Harboring a mutation was also associated with an increase in all-cause mortality that could not be explained by death due to hematological malignancies alone (HR 1.4, 95% CI 1.1-1.8 by Cox proportional hazards model). We further found that these mutations were associated with type 2 diabetes (odds ratio 1.3, 95% CI 1.1-1.5) and increased risk of incident coronary heart disease (HR 2.0, 95% CI 1.2-3.4) and ischemic stroke (HR 2.6, 95% CI 1.4-4.8) in multivariable regression models. We conclude that clonal hematopoiesis associated with a somatic mutation in a known cancer-causing gene is a common pre-malignant condition in the elderly. This entity is associated with increased risk of transformation to hematological malignancy, as well as increased all-cause mortality, possibly due to increased cardio-metabolic disease. While the link between somatic mutations and cancer is well established, the relationship between clonal hematopoiesis and cardio-metabolic disease requires further study. Figure 1 Figure Prevalence of somatic mutation by age. Colored bands represent 50, 75, and 95 percent confidence intervals. Figure. Prevalence of somatic mutation by age. Colored bands represent 50, 75, and 95 percent confidence intervals. Disclosures Getz: The Broad Institute, Inc.: PCT/US2013/057128​ (Detecting Variants in Sequencing Data and Benchmarking Methods and Apparatus for Analyzing and Quantifying DNA Alterations in Cancer)​ Patent pending Patents & Royalties; Appistry: Certain NGS analysis tools of Broad Institute are made available for commercial use via Appistry, Certain NGS analysis tools of Broad Institute are made available for commercial use via Appistry Other. Ebert:Genoptix: Consultancy, Patents & Royalties; Celgene: Consultancy, Research Funding.

Chronic myelogenous leukemia is a myeloproliferative disorder (MPD) that, over time, progresses to acute leukemia. Both processes are closely associated with the t(9;22) chromosomal translocation that creates theBCR/ABLfusion gene in hematopoietic stem cells (HSCs) and their progeny. Chronic myelogenous leukemia is therefore classified as an HSC disorder in which a clone of multipotent HSCs is likely to be malignantly transformed, although direct evidence for malignant t(9;22)+HSCs is lacking. To test whether HSC malignancy is required, we generated hMRP8p210BCR/ABLtransgenic mice in which expression ofBCR/ABLis absent in HSCs and targeted exclusively to myeloid progenitors and their myelomonocytic progeny. Four of 13BCR/ABLtransgenic founders developed a chronic MPD, but only one progressed to blast crisis. To address whether additional oncogenic events are required for progression to acute disease, we crossed hMRP8p210BCR/ABLmice to apoptosis-resistant hMRP8BCL-2mice. Of 18 double-transgenic animals, 9 developed acute myeloid leukemias that were transplantable to wild-type recipients. Taken together, these data indicate that a MPD can arise in mice without expression ofBCR/ABLin HSCs and that additional mutations inhibiting programmed cell death may be critical in the transition of this disease to blast-crisis leukemia.

Human acute myeloid leukemia (AML) is organized as a cellular hierarchy initiated and maintained by rare self-renewing leukemia stem cells (LSC), which must be eliminated in order to cure the patient. We identified increased expression of CD47 on human AML LSC compared to their normal counterparts. CD47 is a cell surface molecule that serves as the ligand for SIRP-alpha on the surface of phagocytes, which in turn transmits a dominant inhibitory signal for phagocytosis. In this way, CD47 essentially functions as a “don't eat me” signal. We hypothesized that increased CD47 expression contributes to pathogenesis by inhibiting phagocytosis of AML LSC. Consistent with this hypothesis, we found that increased CD47 expression predicted worse overall survival in 3 independent cohorts of adult AML patients. Furthermore, we predicted that disruption of the interaction of CD47 with SIRP-alpha would result in phagocytosis and elimination of AML LSC. We found that blocking monoclonal antibodies directed against CD47 enabled phagocytosis of AML LSC, but not normal CD34+ human bone marrow progenitor cells, by human macrophages in vitro. Additionally, coating of human AML LSC with anti-CD47 monoclonal antibodies inhibited their engraftment in vivo in a xenotransplantation assay. Finally, analogous to a clinical therapy, treatment of human AML-engrafted mice with anti-CD47 antibody eliminated AML cells in the peripheral blood and bone marrow and targeted LSC. In summary, increased CD47 expression is an independent poor prognostic factor that can be targeted on human AML stem cells with monoclonal antibodies capable of stimulating phagocytosis and elimination of LSC. Targeting of CD47 with blocking monoclonal antibodies to induce phagocytosis is a novel mechanism for antibody cancer therapy. While anti-CD47 antibodies can be effective monotherapy for human AML, such antibodies may be equally, if not more, effective as part of a combination strategy. The combination of an anti-CD47 antibody, able to block a strong inhibitory signal for phagocytosis, with a second antibody able to bind an LSC-specific molecule and engage Fc receptors on phagocytes, thereby delivering a strong positive signal for phagocytosis, may result in a synergistic stimulus for phagocytosis and specific elimination of AML LSC. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):CN07-03.