Chronic inflammation promotes cancer progression as a second hit

Abstract

•CMO mice (suffering from sterile chronic inflammation) succumb to MLL-AF9 AML faster than WT mice•Hyperactivation of IL-6/Jak/Stat3 in CMO mice contributes to leukemic expansion•CMO Tp53+/− mice show accelerated tumor development compared with that in Tp53+/− mice•CMO Tp53+/− mice exhibit reduced survival and increased risk of AML compared with those in Tp53+/− mice Acute myeloid leukemia (AML) is a malignant neoplasia of the hematopoietic system characterized by the accumulation of immature and nonfunctional leukemic blasts in the bone marrow and peripheral tissues. Mechanistically, the development of AML is explained by the “two-hit” theory, which is based on the accumulation of driver mutations that will cooperate to induce transformation. However, a significant percentage of patients with AML exhibit only one driver mutation, and thus, how leukemic transformation occurs in these cases is unclear. Accumulating evidence suggests that nongenetic factors, such as chronic inflammation, might influence AML development, and accordingly, clinical data have reported that patients with chronic inflammatory disorders have an increased risk of developing hematological malignancies. Here, using a mouse model of chronic inflammation, we demonstrate that systemic elevated levels of cytokines and chemokines and hyperactivation of the Jak/Stat3 signaling pathway may substitute “second hit” mutations and accelerate tumorigenesis. Altogether, our data highlight chronic inflammation as an additional factor in the development of AML, providing additional understanding of the mechanisms of transformation and opening new avenues for the treatment of this disease. Acute myeloid leukemia (AML) is a malignant neoplasia of the hematopoietic system characterized by the accumulation of immature and nonfunctional leukemic blasts in the bone marrow and peripheral tissues. Mechanistically, the development of AML is explained by the “two-hit” theory, which is based on the accumulation of driver mutations that will cooperate to induce transformation. However, a significant percentage of patients with AML exhibit only one driver mutation, and thus, how leukemic transformation occurs in these cases is unclear. Accumulating evidence suggests that nongenetic factors, such as chronic inflammation, might influence AML development, and accordingly, clinical data have reported that patients with chronic inflammatory disorders have an increased risk of developing hematological malignancies. Here, using a mouse model of chronic inflammation, we demonstrate that systemic elevated levels of cytokines and chemokines and hyperactivation of the Jak/Stat3 signaling pathway may substitute “second hit” mutations and accelerate tumorigenesis. Altogether, our data highlight chronic inflammation as an additional factor in the development of AML, providing additional understanding of the mechanisms of transformation and opening new avenues for the treatment of this disease. Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with fast clinical progression and poor prognosis. It is characterized by the expansion of hyperproliferative and dysfunctional myeloid precursors that result from the gradual acquisition of two recurrent driver mutations that provide leukemogenic potential to the cell, as described by the “two-hit” theory [1Kosmider O Moreau-Gachelin F. From mice to human: the “two-hit model” of leukemogenesis.Cell Cycle. 2006; 5: 569-570Crossref PubMed Scopus (13) Google Scholar]. Although each of these mutations alone does not cause leukemic transformation, the co-occurrence of specific abnormalities directly leads to AML development. In accordance with the “two-hit” theory, genomic analysis of 1,540 patients with AML revealed that 86% of them contained two driver mutations [2Papaemmanuil E Gerstung M Bullinger L et al.Genomic classification and prognosis in acute myeloid leukemia.N Engl J Med. 2016; 374: 2209-2221Crossref PubMed Scopus (2798) Google Scholar]. However, how AML developed in the remaining 14% of patients, who harbor only one driver mutation, is unclear. Accumulating evidence suggests that nongenetic factors might as well have a decisive role during AML development [3Eckerling A Ricon-Becker I Sorski L Sandbank E Ben-Eliyahu S. Stress and cancer: mechanisms, significance and future directions.Nat Rev Cancer. 2021; 21: 767-785Crossref PubMed Scopus (110) Google Scholar,4Steensma DP Bejar R Jaiswal S et al.Clonal hematopoiesis of indeterminate potential and its distinction from myelodysplastic syndromes.Blood. 2015; 126: 9-16Crossref PubMed Scopus (1334) Google Scholar]. Interestingly, inflammation has been proposed as a cell external factor that promotes the progression and metastasis of solid tumors, raising a tremendous interest in the role of an inflammatory environment in cancer progression. Accordingly, several population-based studies have identified a history of an autoimmune disease and chronic inflammation as a risk factor for hematological malignancies [5Anderson LA Pfeiffer RM Landgren O Gadalla S Berndt SI Engels EA. Risks of myeloid malignancies in patients with autoimmune conditions.Br J Cancer. 2009; 100: 822-828Crossref PubMed Scopus (209) Google Scholar, 6Cibere J Sibley J Haga M Rheumatoid arthritis and the risk of malignancy.Arthritis Rheum. 1997; 40: 1580-1586Crossref PubMed Scopus (144) Google Scholar, 7Elbæk MV Sørensen AL Hasselbalch HC. Chronic inflammation and autoimmunity as risk factors for the development of chronic myelomonocytic leukemia?.Leuk Lymphoma. 2016; 57: 1793-1799Crossref PubMed Scopus (16) Google Scholar]. Interestingly, recent studies have reported that the presence of inflammation can act as a disease modifier in hematological disorders, that inflammation can be used as a risk stratification factor, and that its modulation can be beneficial in clinical practice [8Ellegast JM Alexe G Hamze A et al.Unleashing cell-intrinsic inflammation as a strategy to kill AML blasts.Cancer Discov. 2022; 12: 1760-1781Crossref PubMed Scopus (12) Google Scholar, 9Lasry A Nadorp B Fornerod M et al.An inflammatory state remodels the immune microenvironment and improves risk stratification in acute myeloid leukemia.Nat Cancer. 2023; 4: 27-42PubMed Google Scholar, 10Mei Y Ren K Liu Y et al.Bone marrow-confined IL-6 signaling mediates the progression of myelodysplastic syndromes to acute myeloid leukemia.J Clin Invest. 2022; 132e152673Crossref Scopus (12) Google Scholar, 11SanMiguel JM Eudy E Loberg MA et al.Distinct tumor necrosis factor alpha receptors dictate stem cell fitness versus lineage output in Dnmt3a-mutant clonal hematopoiesis.Cancer Discov. 2022; 12: 2763-2773Crossref PubMed Scopus (21) Google Scholar]; however, the field still remains largely underexplored. Here, we provide experimental evidence suggesting that elevated systemic levels of proproliferative cytokines associated with chronic inflammation, such as interleukin-6 (IL-6), may provide a proliferative advantage to preleukemic cells through aberrantly activated Jak/Stat3-mediated signaling and, ultimately drive them to AML onset. Please refer to Supplementary Methods. In order to study leukemia development under chronic inflammatory conditions, we employed a mouse model harboring a mutation in the Psptpip2 gene, which causes chronic multifocal osteomyelitis (referred to as CMO mice) [12Drobek A Kralova J Skopcova T et al.PSTPIP2, a protein associated with autoinflammatory disease, interacts with inhibitory enzymes SHIP1 and Csk.J Immunol. 2015; 195: 3416-3426Crossref PubMed Scopus (27) Google Scholar,13Grusanovic S Danek P Kuzmina M et al.Chronic inflammation decreases HSC fitness by activating the druggable Jak/Stat3 signaling pathway.EMBO Rep. 2023; 24: e54729Crossref PubMed Scopus (4) Google Scholar]. CMO is an autoinflammatory disease characterized by elevated systemic levels of proinflammatory cytokines, such as IL-6, IL-1β, MIP-1α, TNFα, and M-CSF [13Grusanovic S Danek P Kuzmina M et al.Chronic inflammation decreases HSC fitness by activating the druggable Jak/Stat3 signaling pathway.EMBO Rep. 2023; 24: e54729Crossref PubMed Scopus (4) Google Scholar,14Chitu V Ferguson PJ de Bruijn R et al.Primed innate immunity leads to autoinflammatory disease in PSTPIP2-deficient cmo mice.Blood. 2009; 114: 2497-2505Crossref PubMed Scopus (64) Google Scholar]. Wild-type (WT) and CMO mice were transplanted with murine leukemic bone marrow (BM) cells isolated from MLL-AF9 primary leukemias (Figure 1A) [15Wang Y Krivtsov AV Sinha AU et al.The Wnt/beta-catenin pathway is required for the development of leukemia stem cells in AML.Science. 2010; 327: 1650-1653Crossref PubMed Scopus (635) Google Scholar]. WT and CMO recipient mice were monitored for any signs of disease, and as expected, all recipient mice developed fatal AML, characterized by excessive amounts of CD11b+ leukemic blasts in the peripheral blood (Figure 1B). However, AML developed significantly faster in CMO mice and showed a more aggressive phenotype (Figure 1C). Accordingly, the percentage of MLL-AF9 GFP+ and c-Kit+ cells was higher in leukemic BM isolated from CMO mice more than in that from WT mice (Figure 1D), suggesting that the inflammatory environment in CMO mice supports a rapid expansion of leukemic cells. Since we recently reported that the inflammatory environment in CMO mice results in the expansion of the hematopoietic stem cell (HSC) pool [13Grusanovic S Danek P Kuzmina M et al.Chronic inflammation decreases HSC fitness by activating the druggable Jak/Stat3 signaling pathway.EMBO Rep. 2023; 24: e54729Crossref PubMed Scopus (4) Google Scholar], we next hypothesized that MLL-AF9 leukemic cells might be expanded in CMO mice through a similar mechanism. Several pieces of evidence suggest that the expansion of HSCs is mediated by the IL-6/Jak/Stat3 signaling pathway in CMO mice. First, CMO mice showed high systemic and BM levels of IL-6 [13Grusanovic S Danek P Kuzmina M et al.Chronic inflammation decreases HSC fitness by activating the druggable Jak/Stat3 signaling pathway.EMBO Rep. 2023; 24: e54729Crossref PubMed Scopus (4) Google Scholar], a proinflammatory cytokine that is produced in large quantities by hematopoietic and nonhematopoietic cells, such as CXCL12-abundant reticular cells, endothelial cells, osteoblasts, myeloid cells, and progenitors, in the BM under stress [16Gerosa RC Boettcher S Kovtonyuk LV et al.CXCL12-abundant reticular cells are the major source of IL-6 upon LPS stimulation and thereby regulate hematopoiesis.Blood Adv. 2021; 5: 5002-5015Crossref PubMed Scopus (10) Google Scholar, 17Ishimi Y Miyaura C Jin CH et al.IL-6 is produced by osteoblasts and induces bone resorption.J Immunol. 1990; 145: 3297-3303Crossref PubMed Google Scholar, 18Schürch CM Riether C Ochsenbein AF. Cytotoxic CD8+ T cells stimulate hematopoietic progenitors by promoting cytokine release from bone marrow mesenchymal stromal cells.Cell Stem Cell. 2014; 14: 460-472Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 19Zhao JL Ma C O'Connell RM et al.Conversion of danger signals into cytokine signals by hematopoietic stem and progenitor cells for regulation of stress-induced hematopoiesis.Cell Stem Cell. 2014; 14: 445-459Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar]. Second, expanded HSCs in CMO mice exhibited an activated IL-6/Jak/Stat3 transcription signature [13Grusanovic S Danek P Kuzmina M et al.Chronic inflammation decreases HSC fitness by activating the druggable Jak/Stat3 signaling pathway.EMBO Rep. 2023; 24: e54729Crossref PubMed Scopus (4) Google Scholar]. In addition, although Stat3 mediates HSC self-renewal [20Chung YJ Park BB Kang YJ Kim TM Eaves CJ Oh IH. Unique effects of Stat3 on the early phase of hematopoietic stem cell regeneration.Blood. 2006; 108: 1208-1215Crossref PubMed Scopus (91) Google Scholar,21Hong SH Yang SJ Kim TM et al.Molecular integration of HoxB4 and STAT3 for self-renewal of hematopoietic stem cells: a model of molecular convergence for stemness.Stem Cells. 2014; 32: 1313-1322Crossref PubMed Scopus (17) Google Scholar], hyperactivation was reported in leukemia [22Cook AM Li L Ho Y et al.Role of altered growth factor receptor-mediated JAK2 signaling in growth and maintenance of human acute myeloid leukemia stem cells.Blood. 2014; 123: 2826-2837Crossref PubMed Scopus (87) Google Scholar,23Vainchenker W Constantinescu SN. JAK/STAT signaling in hematological malignancies.Oncogene. 2013; 32: 2601-2613Crossref PubMed Scopus (419) Google Scholar]. These data prompted us to assess the levels of pStat3 in MLL-AF9 leukemic cells isolated from WT and CMO mice. We observed that pStat3 levels were increased in leukemic BM cells isolated from CMO mice (Figure 1E), suggesting that hyperactivation of this pathway could contribute to accelerated leukemic development. Next, we investigated the potential role of IL-6 in Stat3 activation in MLL-AF9 cells and observed increased pStat3 levels in MLL-AF9 cells upon IL-6 treatment compared with those in nontreated control cells (Figure 2A). Furthermore, we seeded MLL-AF9 leukemic BM cells in a semisolid medium supplemented with 1 ng/mL of IL-3 and various concentrations of IL-6. Indeed, the number of colony-forming units (CFUs), reflecting the presence of the leukemic stem and progenitor cells in the culture, was significantly increased by IL-6 treatment (Figure 2B). In addition, the colonies increased in size, as demonstrated by the increased cell numbers, in an IL-6 dose-dependent manner (Figure 2C). Although cell survival was not changed in the IL-6–containing cultures, we observed enhanced cell proliferation as the concentration of IL-6 in the culture was increased (Figure 2D and E). Furthermore, we assessed whether IL-6 inhibition in vivo would be sufficient to slow down the leukemic progression in the MLL-AF9 CMO model (Supplementary Figure E1A). We observed that treatment with an IL-6 neutralizing antibody did not affect leukemic progression (Supplementary Figure E1B and C), suggesting that other cytokines may contribute to the activation of Stat3 in leukemic CMO mice. Consistently, cultivation of MLL-AF9 leukemic BM cells with various concentrations of Stattic, a Stat3 inhibitor, dramatically reduced CFU numbers, whereas control WT BM remained unaffected (Figure 2F and G). Therefore, we investigated whether Stat3 inhibition would have a similar effect in vivo (Figure 2H). Despite not observing an increased lifespan of MLL-AF9 CMO recipients who received Stattic treatment (Figure 2I), we noticed that this group of mice had reduced MLL-AF9 leukemic burden 3 weeks after transplantation and treatment (Figure 2J). We speculate that given the aggressive nature of MLL-AF9 AML, Stattic treatment was not sufficient to extend the survival of CMO mice despite the fact that the expansion of leukemic cells could be moderated during the treatment. Altogether, our results suggest that chronic inflammation promotes the expansion of leukemic cells through hyperactivation of the Jak/Stat3 signaling pathway, pointing at the contribution of IL-6 and suggesting the participation of additional cytokines. We showed that the inflammatory environment supports the expansion of MLL-AF9 leukemic cells and enhances the severity of the disease. Nevertheless, our data so far do not clarify whether inflammation, by itself, can mediate the transition from preleukemic cells to fully transformed leukemic cells. To answer this question, we combined the CMO environment with a suitable preleukemic model. Loss-of-function mutations of the TP53 tumor suppressor gene were identified as an early event in approximately 15% of newly diagnosed patients with AML [24Lal R Lind K Heitzer E et al.Somatic TP53 mutations characterize preleukemic stem cells in acute myeloid leukemia.Blood. 2017; 129: 2587-2591Crossref PubMed Scopus (41) Google Scholar,25Lindsley RC Mar BG Mazzola E et al.Acute myeloid leukemia ontogeny is defined by distinct somatic mutations.Blood. 2015; 125: 1367-1376Crossref PubMed Scopus (692) Google Scholar]. However, leukemia development was not observed in TP53-deficient mice, although they spontaneously developed several types of solid tumors [26Donehower LA Harvey M Slagle BL et al.Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours.Nature. 1992; 356: 215-221Crossref PubMed Scopus (4222) Google Scholar]. Therefore, we crossed homozygous (Tp53−/−) and heterozygous (Tp53+/−) Tp53-deficient mice with CMO mice and monitored their survival (Figure 3A). As expected, homozygous Tp53−/− developed fatal tumors with a median survival of 141 days, while the mortality of Tp53+/− heterozygotes was delayed (median survival, 201 days). The combination of CMO inflammatory environment with homozygous Tp53 deficiency resulted in a trend of reduced survival of CMO Tp53−/− double mutants compared with single Tp53−/− mutants (p = 0.1023). Remarkably, heterozygous CMO Tp53+/− double mutants showed significantly accelerated tumor development and reduced survival compared with their Tp53+/− noninflammatory counterpart (Figure 3A). Importantly, in a follow-up cohort, one of 20 CMO Tp53+/− mice developed myeloid leukemia, represented by splenomegaly, tumor mass detected in the liver and spleen, and increased percentage of c-Kit and myeloid cells in the BM and spleen (Figure 3B–D). Furthermore, CMO Tp53+/− exhibited larger spleens than single mutant Tp53+/− mice, while other types of tumors were observed in both CMO Tp53+/− and Tp53+/− mice (Figure 3E and F and Supplementary Table E1). These results suggest that the sole presence of chronic inflammation can accelerate tumor development and act as a leukemogenic driving factor, potentially increasing the risk of leukemia development, specifically in patients with clinically relevant TP53 mutations. In line with our observations, it has recently been reported that chronic inflammation acts as a driver in TP53-mutant leukemia in human patients [27Rodriguez-Meira A Norfo R Wen S et al.Single-cell multi-omics identifies chronic inflammation as a driver of TP53-mutant leukemic evolution.Nat Genet. 2023; 55: 1531-1541Crossref PubMed Scopus (12) Google Scholar]. A limitation of our study is that we observed only one leukemia case in our CMO Tp53+/− cohort (n = 20). However, the stochasticity and low probability of the occurrence of de novo driver mutations together with the short lifespan of the mice might provide an explanation. Future experiments should determine whether transplantation of Tp53+/− or Tp53−/− cells into WT and CMO recipients, instead of genetic crossing, would affect the kinetics of transformation and/or the type of hematological malignancy. The results could consolidate the role of a proinflammatory environment in the development of myeloid rather than lymphoid leukemias. In addition, future analysis of our murine models should include whole exome sequencing to determine the possible acquisition of somatic mutations under chronic inflammation conditions. This experiment would clarify whether chronic inflammation has a direct effect on leukemia progression by merely promoting proliferation of leukemic cells or whether chronic inflammation might induce DNA damage and, thus, additional oncogenic mutations. Of note, although mutations in Pstpip2 have not been described as driver mutations in leukemia or any other cancer, we need to keep in mind that our murine models harbor an additional mutation because the Pstpip2 mutation is present in all tissues in the Tp53 model and in CMO recipient mice in the MLL-AF9 model. Nonetheless, this brief study provides proof-of-concept results showing that increased levels of proinflammatory cytokines, such as IL-6, during chronic inflammation might result in an aberrant activation of Stat3 signaling and promote expansion of leukemic cells. When combined with other driver mutations, such as TP53, this augmented expansion might act as a critical factor necessary for premalignant cells to transition to malignant cells. Therefore, the current “two-hit” model of leukemogenesis is not complete and should also include nongenetic factors, such as chronic inflammation (Figure 3G). The authors do not have any conflicts of interest to declare in relation to this work. The study was supported by the Czech Science Foundation GACR grant 20-03380S, by the National Institute for Cancer Research (Program EXCELES, ID Project number LX22NPO5102)—funded by the European Union—Next Generation EU, and by institutional funding from the Institute of Molecular Genetics of the Czech Academy of Sciences (IMG CAS) (RVO 68378050) (to Dr. M Alberich-Jorda). This project received funding from the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement number 860002. The flow cytometry data presented in this paper were produced at the Flow Cytometry Core Facility, IMG CAS, Prague, Czech Republic, and histology data were prepared and analyzed by the Czech Centre for Phenogenomics of the IMG CAS. We thank Zuzana Chalupova for technical assistance. MA-J conceived and designed the study; MB, and and MA-J data analysis and and MA-J the The in the will be to any to them for and after by the with with with

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