An inflammatory environment containing TNFα favors Tet2-mutant clonal hematopoiesis

•Inactivating TET2 mutations are common drivers of clonal hematopoiesis with aging.•Tumor necrosis factor-alpha (TNFα) is increased in myelodysplastic syndromes (MDS) and unhealthy aging.•Loss of Tet2 increases murine bone marrow (BM) colony formation in the presence of TNFα.•Human TET2-mutant BM is resistant to colony-suppressive effects of TNFα.•TET2-mutant clones may have greater fitness in an inflammatory environment of aging. Clonal hematopoiesis of aging and indeterminate potential (ARCH or CHIP), driven mainly by mutations in DNMT3A and TET2, is an emerging public health issue, affecting at least 10–15% of adults older than 65 years. CHIP is associated with increased risks of de novo and therapy-related hematological neoplasms and serves as a reservoir for leukemic relapse. CHIP is also associated with increased all-cause mortality and risk of cardio-metabolic disease. The latter association may be explained, at least in part, by the effects of inactivating mutations in TET2 on progeny macrophages. We and others have shown recently that TET2-deficient macrophages are hyperinflammatory and this may exacerbate processes such as atherosclerosis. We postulated an inflammatory state associated with TET2 inactivation and/or unhealthy aging may also favor TET2-mutant hematopoietic stem and progenitor cell (HSPC) expansion. Herein, we demonstrate a clonogenic advantage for Tet2-knockout murine and TET2-mutant human HSPCs in an in vitro environment that contains the proinflammatory cytokine tumor necrosis factor-alpha (TNFα). This phenotype emerges on chronic TNFα exposure and is associated with myeloid skewing and resistance to apoptosis. To our knowledge, this is the first evidence to suggest that TET2 mutations promote clonal dominance with aging by conferring TNFα resistance to sensitive bone marrow progenitors while also propagating such an inflammatory environment. Normalizing the immune environment may present a novel strategy to control or eradicate mutant CHIP clones. Clonal hematopoiesis of aging and indeterminate potential (ARCH or CHIP), driven mainly by mutations in DNMT3A and TET2, is an emerging public health issue, affecting at least 10–15% of adults older than 65 years. CHIP is associated with increased risks of de novo and therapy-related hematological neoplasms and serves as a reservoir for leukemic relapse. CHIP is also associated with increased all-cause mortality and risk of cardio-metabolic disease. The latter association may be explained, at least in part, by the effects of inactivating mutations in TET2 on progeny macrophages. We and others have shown recently that TET2-deficient macrophages are hyperinflammatory and this may exacerbate processes such as atherosclerosis. We postulated an inflammatory state associated with TET2 inactivation and/or unhealthy aging may also favor TET2-mutant hematopoietic stem and progenitor cell (HSPC) expansion. Herein, we demonstrate a clonogenic advantage for Tet2-knockout murine and TET2-mutant human HSPCs in an in vitro environment that contains the proinflammatory cytokine tumor necrosis factor-alpha (TNFα). This phenotype emerges on chronic TNFα exposure and is associated with myeloid skewing and resistance to apoptosis. To our knowledge, this is the first evidence to suggest that TET2 mutations promote clonal dominance with aging by conferring TNFα resistance to sensitive bone marrow progenitors while also propagating such an inflammatory environment. Normalizing the immune environment may present a novel strategy to control or eradicate mutant CHIP clones. Inactivating mutations of tet methylcytosine dioxygenase 2 (TET2) are among the earliest and most common in clonal hematopoiesis associated with aging (CHIP) and myeloid neoplasms, including myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML) [1Jaiswal S. Fontanillas P. Flannick J. et al.Age-related clonal hematopoiesis associated with adverse outcomes.N Engl J Med. 2014; 371: 2488-2498Crossref PubMed Scopus (2506) Google Scholar, 2Genovese G. Kähler A.K. Handsaker R.E. et al.Clonal hematopoiesis and blood-cancer risk inferred from blood DNA sequence.N Engl J Med. 2014; 371: 2477-2487Crossref PubMed Scopus (1978) Google Scholar, 3Zink F. Stacey S.N. Norddahl G.L. et al.Clonal hematopoiesis, with and without candidate driver mutations, is common in the elderly.Blood. 2017; 130: 742-752Crossref PubMed Scopus (400) Google Scholar, 4Buscarlet M. Provost S. Feroz Zada Y. et al.DNMT3A and TET2 dominate clonal hematopoiesis, demonstrate benign phenotypes and different genetic predisposition.Blood. 2017; 130: 753-762Crossref PubMed Scopus (215) Google Scholar, 5Delhommeau F. Dupont S. Della Valle V. et al.Mutation in TET2 in myeloid cancers.N Engl J Med. 2009; 360: 2289-2301Crossref PubMed Scopus (1405) Google Scholar, 6Papaemmanuil E. Gerstung M. Malcovati L. et al.Chronic Myeloid Disorders Working Group of the International Cancer Genome ConsortiumClinical and biological implications of driver mutations in myelodysplastic syndromes.Blood. 2013; 122: 3616-3627Crossref PubMed Scopus (1256) Google Scholar]. The encoded protein, TET2, is an epigenetic regulator that catalyzes the conversion of 5-methylcytosine to 5-hydroxymethylcytosine as the first step in cytosine demethylation [7Ko M. Huang Y. Jankowska A.M. et al.Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2.Nature. 2010; 468: 839-843Crossref PubMed Scopus (1023) Google Scholar]. Inactivation of TET2 in human (and Tet2 in murine) hematopoietic precursors leads to a stem cell advantage with skewed differentiation to myelomonocytic/macrophage lineages and Tet2-deficient murine models phenocopy human MDS and CMML [5Delhommeau F. Dupont S. Della Valle V. et al.Mutation in TET2 in myeloid cancers.N Engl J Med. 2009; 360: 2289-2301Crossref PubMed Scopus (1405) Google Scholar, 7Ko M. Huang Y. Jankowska A.M. et al.Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2.Nature. 2010; 468: 839-843Crossref PubMed Scopus (1023) Google Scholar, 8Moran-Crusio K. Reavie L. Shih A. et al.Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation.Cancer Cell. 2011; 20: 11-24Abstract Full Text Full Text PDF PubMed Scopus (931) Google Scholar, 9Quivoron C. Couronné L. Della Valle V. et al.TET2 inactivation results in pleiotropic hematopoietic abnormalities in mouse and is a recurrent event during human lymphomagenesis.Cancer Cell. 2011; 20: 25-38Abstract Full Text Full Text PDF PubMed Scopus (648) Google Scholar, 10Li Z. Cai X. Cai C.L. et al.Deletion of Tet2 in mice leads to dysregulated hematopoietic stem cells and subsequent development of myeloid malignancies.Blood. 2011; 118: 4509-4518Crossref PubMed Scopus (493) Google Scholar]. It remains to be determined, however, why the emergence of TET2-mutant (and other) clones is associated with increasing age [1Jaiswal S. Fontanillas P. Flannick J. et al.Age-related clonal hematopoiesis associated with adverse outcomes.N Engl J Med. 2014; 371: 2488-2498Crossref PubMed Scopus (2506) Google Scholar, 2Genovese G. Kähler A.K. Handsaker R.E. et al.Clonal hematopoiesis and blood-cancer risk inferred from blood DNA sequence.N Engl J Med. 2014; 371: 2477-2487Crossref PubMed Scopus (1978) Google Scholar, 3Zink F. Stacey S.N. Norddahl G.L. et al.Clonal hematopoiesis, with and without candidate driver mutations, is common in the elderly.Blood. 2017; 130: 742-752Crossref PubMed Scopus (400) Google Scholar, 4Buscarlet M. Provost S. Feroz Zada Y. et al.DNMT3A and TET2 dominate clonal hematopoiesis, demonstrate benign phenotypes and different genetic predisposition.Blood. 2017; 130: 753-762Crossref PubMed Scopus (215) Google Scholar]. It has been proposed that selective pressures associated with an aging hematopoietic system, and perhaps inflammation, favors the expansion of fitter mutants [11McKerrell T. Vassiliou G.S. Aging as a driver of leukemogenesis.Sci Transl Med. 2015; 7: 306fs38Crossref PubMed Scopus (29) Google Scholar, 12Shlush L.I. Zandi S. Itzkovitz S. Schuh A.C. Aging, clonal hematopoiesis and preleukemia: not just bad luck?.Int J Hematol. 2015; 102: 513-522Crossref PubMed Scopus (23) Google Scholar]; however, direct evidence is lacking. We and others have demonstrated that Tet2-mutant progeny macrophages are hyperinflammatory, a phenotype that may contribute to comorbidities such as cardiovascular disease [13Cull A.H. Snetsinger B. Buckstein R. Wells R.A. Rauh M.J. Tet2 restrains inflammatory gene expression in macrophages.Exp Hematol. 2017; 55 (e13): 56-70Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 14Zhang Q. Zhao K. Shen Q. et al.Tet2 is required to resolve inflammation by recruiting Hdac2 to specifically repress IL-6.Nature. 2015; 525: 389-393Crossref PubMed Scopus (459) Google Scholar, 15Fuster J.J. MacLauchlan S. Zuriaga M.A. et al.Clonal hematopoiesis associated with TET2 deficiency accelerates atherosclerosis development in mice.Science. 2017; 355: 842-847Crossref PubMed Scopus (686) Google Scholar, 16Jaiswal S. Natarajan P. Silver A.J. et al.Clonal hematopoiesis and risk of atherosclerotic cardiovascular disease.N Engl J Med. 2017; 377: 111-121Crossref PubMed Scopus (1117) Google Scholar]. We therefore hypothesized that Tet2-mutant hematopoietic stem/progenitor cells (HSPCs) would demonstrate greater fitness in an environment that contains cytokines associated with MDS and “inflamm-aging” (i.e., tumor necrosis factor-alpha [TNFα]) [17Navas T.A. Mohindru M. Estes M. et al.Inhibition of overactivated p38 MAPK can restore hematopoiesis in myelodysplastic syndrome progenitors.Blood. 2006; 108: 4170-4177Crossref PubMed Scopus (106) Google Scholar, 18Franceschi C. Bonafè M. Valensin S. et al.Inflamm-aging. An evolutionary perspective on immunosenescence.Ann N Y Acad Sci. 2000; 908: 244-254Crossref PubMed Scopus (3139) Google Scholar]. Adult (10–14 weeks old) Tet2 wild-type and Tet2-mutant C57BL/6 mice (JAX) were chosen as a model system. The floxed Tet2 allele (Tet2f) was deleted by targeting exon 3 with Vav1-cre mediated, hematopoietic-directed excision [8Moran-Crusio K. Reavie L. Shih A. et al.Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation.Cancer Cell. 2011; 20: 11-24Abstract Full Text Full Text PDF PubMed Scopus (931) Google Scholar]. Vav1-cre transgenic mice were bred with Tet2f/f mice to create homozygous Tet2-knockouts (Vav1-cre+;Tet2−/−, i.e. Tet2−/−) and controls (Vav-cre-;Tet2f/f, i.e. Tet2f/f or wild-type, wt). By 10–14 weeks of age, Tet2−/− mice begin to manifest features of cytopenia, monocytosis, splenomegaly (CMML) and this becomes more pronounced by 20 weeks [8Moran-Crusio K. Reavie L. Shih A. et al.Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation.Cancer Cell. 2011; 20: 11-24Abstract Full Text Full Text PDF PubMed Scopus (931) Google Scholar]. We isolated lineage-negative cells (Lin−) enriched for HSPCs from wt and Tet2−/− bone marrow (BM) (EasySep; StemCell Technologies) and cultured these in the absence or presence of TNFα (0.1, 1, or 10 ng/mL) in a standard methylcellulose colony formation assay formulation (MethoCult M3434; StemCell Technologies) or liquid culture medium and then examined colony growth characteristics and apoptosis over 12 days. Where indicated, serial replating was performed to simulate prolonged cytokine exposure. We also examined colony growth of TET2-mutant and nonmutant human MDS under varying TNFα concentrations using frozen BM mononuclear cells (BMMNCs) from MDS patients and healthy controls. Murine and human studies were approved by the Queen's University Animal Care Committee and human ethics boards of Queen's University and Sunnybrook Health Sciences Centre, respectively. Total RNA was harvested (Qiagen) and subjected to SYBR Green qRT-PCR (Thermo Fisher) to measure relative levels of Tnf receptor superfamily member1a (Tnfrsf1a), 1b (Tnfrsf1b), Fas cell surface death receptor (Fas), Caspase 3 (Casp3), Caspase 8 (Casp8), Bcl2 apoptosis regulator (Bcl2), Baculoviral IAP repeat containing 2 (Birc2), and Bcl2 like 1 (Bcl2l1) using Hydroxymethylbilane synthase (Hmbs) as reference mRNA. Forward and reverse primers, respectively, were as follows: Tnfrsf1a: GGGCACCTTTACGGCTTCC, GGTTCTCCTTACAGCCACACA; Tnfrsf1b: CAGGTTGTCTTGACACCCTAC, GCACAGCACATCTGAGCCT; Fas: TATCAAGGAGGCCCATTTTGC, TGTTTCCACTTCTAAACCATGCT; Casp3: AAGATCATAGCAAAAGGAGCAG, GAGTAAGCATACAGGAAGTCAG; Casp8: AAGAACTGGGCAGTGA, TCTAGGAAGTTGACCA; Bcl2: GTACCTGAACCGGCATCTG, GGGGCCATATAGTTCCACAA; Birc2: GTTTTAAAACCAGCTTGGGTTATATTG, GTTCCTCACCCAACCAGTCTACTTAG; Bcl2l1: TGGAGTAAACTGGGGGTCGCATCG, AGCCACCGTCATGCCCGTCAGG; and Hmbs: GAGTCTAGATGGCTCAGATAGCATGC, CCTACAGACCAGTTAGCGCACATC. Recently, we reported increased plasma levels of TNFα in Tet2−/− versus wt mice [13Cull A.H. Snetsinger B. Buckstein R. Wells R.A. Rauh M.J. Tet2 restrains inflammatory gene expression in macrophages.Exp Hematol. 2017; 55 (e13): 56-70Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar] and found 7.6-fold higher mean Tnf mRNA levels in Tet2−/− versus wt Lin− BM cells by qRT-PCR. Therefore, we first assessed the effects of TNFα on wt and Tet2−/− Lin− BM cells using in vitro colony-forming assays (104 Lin− cells/well). TNFα is known to exert suppressive effects on HSPC activity [17Navas T.A. Mohindru M. Estes M. et al.Inhibition of overactivated p38 MAPK can restore hematopoiesis in myelodysplastic syndrome progenitors.Blood. 2006; 108: 4170-4177Crossref PubMed Scopus (106) Google Scholar]; consistent with this, we observed a dose-dependent reduction in colony counts in both genotypes on days 3, 6, 9, and 12 (3-fold for wt and 2.5-fold for −/− at 0 vs. 10 ng/mL, day 12, p = 0.0097 and p = 0.0145, respectively) (Fig. 1A). It has been reported previously that Tet2-deficient HSPCs have an enhanced ability to serially replate and generate new colonies compared with wt [8Moran-Crusio K. Reavie L. Shih A. et al.Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation.Cancer Cell. 2011; 20: 11-24Abstract Full Text Full Text PDF PubMed Scopus (931) Google Scholar]. To determine whether this phenomenon holds true under the suppressive pressure of TNFα and to mimic chronic inflammatory exposure, we harvested cells from the semi-solid medium after 12 days and replated them under increasing concentrations of TNFα. In the presence of TNFα, Tet2−/− BM cells demonstrated a striking replating advantage. As exemplified by day 12 colony counts at first replating (Fig. 1B) and subsequent serial replating (not shown), whereas wt colonies again declined with increasing TNFα, Tet2−/− counts were at least maintained or even increased with TNFα concentration (mean 19 colonies at replating day 12 for Tet2−/− at 1 ng/mL vs. 11.75 at 0 ng/mL). Moreover, similar to Tet2−/− cells, haplo-insufficient Tet2+/− cells displayed superior replating capacity compared with Tet2f/+ and Tet2f/f controls under prolonged TNFα stress (not shown). To demonstrate that this phenotype was not unique to semisolid media, we cultured Tet2−/− and wt Lin− BM cells under long-term culture conditions (Iscove modified Dulbecco medium supplemented with 100 ng/mL stem cell factor, 20 ng/mL interleukin-6, and 10 ng/mL thrombopoietin) ± TNFα . Similar to semisolid colony assays, both Tet2−/− and wt Lin− BM cells were initially sensitive to the growth-suppressive effect of TNFα (Figs. 1C and 1D). However, after a media change at day 15, an expanding population appeared in Tet2−/− cultures that showed significantly increased resistance to TNFα (Fig. 1D). Taken together, these observations demonstrated resistance of Tet2−/− HSPCs to the suppressive effects of TNFα upon chronic exposure. To gain mechanistic insight into this resistant phenotype, we chose to focus on Tet2−/− and wt responses to TNFα. We first sought additional information about the nature of the baseline and emergent colonies. Consistent with previous reports [8Moran-Crusio K. Reavie L. Shih A. et al.Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation.Cancer Cell. 2011; 20: 11-24Abstract Full Text Full Text PDF PubMed Scopus (931) Google Scholar, 9Quivoron C. Couronné L. Della Valle V. et al.TET2 inactivation results in pleiotropic hematopoietic abnormalities in mouse and is a recurrent event during human lymphomagenesis.Cancer Cell. 2011; 20: 25-38Abstract Full Text Full Text PDF PubMed Scopus (648) Google Scholar, 10Li Z. Cai X. Cai C.L. et al.Deletion of Tet2 in mice leads to dysregulated hematopoietic stem cells and subsequent development of myeloid malignancies.Blood. 2011; 118: 4509-4518Crossref PubMed Scopus (493) Google Scholar], Tet2−/− HSPCs produced more granulocyte, erythrocyte, monocyte, megakaryocyte colony-forming units (CFU-GEMM) than wt at day 12 of first plating and a reciprocal trend to reduced erythrocyte burst-forming units (BFU-E) (Fig. 1E). Moreover, TNFα reduced mean CFU-GEMM and BFU-E colony counts in both genotypes, but only granulocyte-macrophage colony-forming units (CFU-GM) in wt (Fig. 1E). Upon replating, these differences between genotypes were exaggerated, with Tet2−/− GEMM colonies being resistant to TNFα suppression and Tet2−/− GM colonies paradoxically increasing (Fig. 1F). These findings suggested that chronic inflammatory cytokine exposure provided a permissive environment for clonal dominance of Tet2-mutant HSPCs with an associated myeloid bias. Next, we found Tet2−/− colonies contained more cells per colony under TNFα stress (not shown), suggesting increased survival and/or proliferation. Consistent with this, using Annexin V–propidium iodide flow cytometry, we showed that whereas both genotypes were initially sensitive to TNFα-induced apoptosis, Tet2−/− BM cells had a lower apoptotic index compared with wt with chronic exposure to TNFα in vitro (Figs. 2A and 2B). Next, we measured mRNA expression levels (using SYBR green qRT-PCR) of the proapoptotic targets TNFRI (Tnfrsf1a), TNFRII (Tnfrsf1b), Fas, caspase 3 (Casp3), and Casp8 and the antiapoptotic targets Bcl2, Bcl-XL (Bcl2l1), and IAP1 (Birc2) in both wt and Tet2−/− progenitor cells relative to Hbms after chronic TNFα exposure (i.e. at the end of replating on day 12). Consistent with a gene expression profile mediating resistance to apoptosis, the mRNA levels of Tnfrsf1a, Tnfrsf1b, Fas, Casp3, and Casp8 were significantly lower in Tet2−/−cells compared with wt under inflammatory stress of TNFα (Figs. 2C–2G), whereas Bcl2 and Birc2 were significantly elevated (Figs. 2H and 2I). The correlation was not perfect, however, because expression of anti-apoptotic Bcl2l1 was decreased in Tet2−/− (Fig. 2J). To determine whether these murine findings translated to humans, we also examined semisolid colony growth of TET2-mutant and nonmutant BM on in vitro exposure to varying TNFα concentrations using frozen BMMNCs from MDS patients and healthy controls. At first exposure, BFU-E and CFU-GM colonies in TET2-mutant MDS were resistant to TNFα inhibition compared with wt and controls (Figs. 3A and 3B). To our knowledge, this is the first in vitro evidence to suggest that Tet2/TET2 mutations promote clonal dominance with aging by conferring TNFα resistance to sensitive BM progenitors while also propagating such an inflammatory environment [13Cull A.H. Snetsinger B. Buckstein R. Wells R.A. Rauh M.J. Tet2 restrains inflammatory gene expression in macrophages.Exp Hematol. 2017; 55 (e13): 56-70Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 14Zhang Q. Zhao K. Shen Q. et al.Tet2 is required to resolve inflammation by recruiting Hdac2 to specifically repress IL-6.Nature. 2015; 525: 389-393Crossref PubMed Scopus (459) Google Scholar, 15Fuster J.J. MacLauchlan S. Zuriaga M.A. et al.Clonal hematopoiesis associated with TET2 deficiency accelerates atherosclerosis development in mice.Science. 2017; 355: 842-847Crossref PubMed Scopus (686) Google Scholar, 16Jaiswal S. Natarajan P. Silver A.J. et al.Clonal hematopoiesis and risk of atherosclerotic cardiovascular disease.N Engl J Med. 2017; 377: 111-121Crossref PubMed Scopus (1117) Google Scholar]. Formal support of the in vivo relevance will require competitive hematopoietic stem cell repopulation experiments in lethally irradiated mice with genetic and/or pharmacological manipulation of TNFα expression or activity and these studies are planned. Mutations that promote resistance to environmental stem cell stressors are a known mechanism of clonal selection in Fanconi anemia [19Li J. Sejas D.P. Zhang X. et al.TNF-alpha induces leukemic clonal evolution ex vivo in Fanconi anemia group C murine stem cells.J Clin Invest. 2007; 117: 3283-3295Crossref PubMed Scopus (109) Google Scholar], chronic myeloid leukemia [20Reynaud D. Pietras E. Barry-Holson K. et al.IL-6 controls leukemic multipotent progenitor cell fate and contributes to chronic myelogenous leukemia development.Cancer Cell. 2011; 20: 661-673Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar], and JAK2-mutant myeloproliferative neoplasms [21Fleischman A.G. Aichberger K.J. Luty S.B. et al.TNFα facilitates clonal expansion of JAK2V617F positive cells in myeloproliferative neoplasms.Blood. 2011; 118: 6392-6398Crossref PubMed Scopus (187) Google Scholar]. Given that inflammatory cytokines have been associated with inflamm-aging and myelodysplasia [17Navas T.A. Mohindru M. Estes M. et al.Inhibition of overactivated p38 MAPK can restore hematopoiesis in myelodysplastic syndrome progenitors.Blood. 2006; 108: 4170-4177Crossref PubMed Scopus (106) Google Scholar, 18Franceschi C. Bonafè M. Valensin S. et al.Inflamm-aging. An evolutionary perspective on immunosenescence.Ann N Y Acad Sci. 2000; 908: 244-254Crossref PubMed Scopus (3139) Google Scholar], it is worth exploring further whether TET2-mutant human clones may emerge under inflammatory stress with enhanced resistance to apoptosis, leading to CHIP and myeloid cancers such as MDS and presenting a novel therapeutic target for clone eradication [22Cimmino L. Dolgalev I. Wang Y. et al.Restoration of TET2 function blocks aberrant self-renewal and leukemia progression.Cell. 2017; 170 (e20): 1079-1095Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar, 23Agathocleous M. Meacham C.E. Burgess R.J. et al.Ascorbate regulates haematopoietic stem cell function and leukaemogenesis.Nature. 2017; 549: 476-481Crossref PubMed Scopus (303) Google Scholar]. We thank Brooke Snetsinger for technical assistance, the Queen's University Animal Care facility staff, and Lilly Gu for collecting and shipping bone marrow samples. This work was funded by research grants awarded to MJR from Queen's University (Kingston, Ontario), the Canada Foundation for Innovation (Ottawa, Ontario), the Ontario Institute for Cancer Research (Toronto, Ontario), the Southeastern Ontario Academic Medical Organization (Kingston, Ontario), and the Women's Giving Circle/University Hospitals Kingston Foundation (Kingston, Ontario). SOA was supported in part by the Queen's University Terry Fox Foundation Training Program in Transdisciplinary Cancer Research in partnership with the Canadian Institutes of Health Research.