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• W2521164667 abstract "•Neither primary B-ALL blasts nor leukemic B cell lines can be reprogrammed to iPSCs•Global transcriptome and DNA methylome suggest a developmental refractoriness Induced pluripotent stem cells (iPSCs) are a powerful tool for disease modeling. They are routinely generated from healthy donors and patients from multiple cell types at different developmental stages. However, reprogramming leukemias is an extremely inefficient process. Few studies generated iPSCs from primary chronic myeloid leukemias, but iPSC generation from acute myeloid or lymphoid leukemias (ALL) has not been achieved. We attempted to generate iPSCs from different subtypes of B-ALL to address the developmental impact of leukemic fusion genes. OKSM(L)-expressing mono/polycistronic-, retroviral/lentiviral/episomal-, and Sendai virus vector-based reprogramming strategies failed to render iPSCs in vitro and in vivo. Addition of transcriptomic-epigenetic reprogramming “boosters” also failed to generate iPSCs from B cell blasts and B-ALL lines, and when iPSCs emerged they lacked leukemic fusion genes, demonstrating non-leukemic myeloid origin. Conversely, MLL-AF4-overexpressing hematopoietic stem cells/B progenitors were successfully reprogrammed, indicating that B cell origin and leukemic fusion gene were not reprogramming barriers. Global transcriptome/DNA methylome profiling suggested a developmental/differentiation refractoriness of MLL-rearranged B-ALL to reprogramming into pluripotency. Induced pluripotent stem cells (iPSCs) are a powerful tool for disease modeling. They are routinely generated from healthy donors and patients from multiple cell types at different developmental stages. However, reprogramming leukemias is an extremely inefficient process. Few studies generated iPSCs from primary chronic myeloid leukemias, but iPSC generation from acute myeloid or lymphoid leukemias (ALL) has not been achieved. We attempted to generate iPSCs from different subtypes of B-ALL to address the developmental impact of leukemic fusion genes. OKSM(L)-expressing mono/polycistronic-, retroviral/lentiviral/episomal-, and Sendai virus vector-based reprogramming strategies failed to render iPSCs in vitro and in vivo. Addition of transcriptomic-epigenetic reprogramming “boosters” also failed to generate iPSCs from B cell blasts and B-ALL lines, and when iPSCs emerged they lacked leukemic fusion genes, demonstrating non-leukemic myeloid origin. Conversely, MLL-AF4-overexpressing hematopoietic stem cells/B progenitors were successfully reprogrammed, indicating that B cell origin and leukemic fusion gene were not reprogramming barriers. Global transcriptome/DNA methylome profiling suggested a developmental/differentiation refractoriness of MLL-rearranged B-ALL to reprogramming into pluripotency. Leukemia is generally studied once the full transformation events have already occurred and, therefore, the mechanisms by which leukemia-specific mutations transform to a pre-leukemic state followed by rapid transition to overt leukemia are not amenable to analysis with patient samples (Ramos-Mejia et al., 2012cRamos-Mejia V. Fraga M.F. Menendez P. iPSCs from cancer cells: challenges and opportunities.Trends Mol. Med. 2012; 18: 245-247Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Therefore, it is imperative to develop effective disease models to study the developmental impact of leukemia-specific genetic aberrations on human stem cell fate. Induced pluripotent stem cells (iPSCs) are a powerful tool for modeling different aspects of human disease that cannot otherwise be addressed by patient sample analyses or animal models (Menendez et al., 2006Menendez P. Bueno C. Wang L. Human embryonic stem cells: a journey beyond cell replacement therapies.Cytotherapy. 2006; 8: 530-541Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, Wu and Hochedlinger, 2011Wu S.M. Hochedlinger K. Harnessing the potential of induced pluripotent stem cells for regenerative medicine.Nat. Cell Biol. 2011; 13: 497-505Crossref PubMed Scopus (405) Google Scholar). Because leukemia manifests as a developmental cell blockage, the generation and differentiation of leukemia-specific iPSCs offers a promising strategy to study the earliest events leading to the specification of both normal and abnormal hematopoietic tissue, thus illuminating molecular mechanisms underlying the pathogenesis of human leukemia. iPSCs are routinely generated from tissues obtained from healthy donors and patients and cell types at different developmental stages. Reprogramming human primary cancer cells, however, remains challenging. Despite significant interest in generating iPSCs from leukemia cells (Curry et al., 2015Curry E.L. Moad M. Robson C.N. Heer R. Using induced pluripotent stem cells as a tool for modelling carcinogenesis.World J. Stem Cells. 2015; 7: 461-469Crossref PubMed Google Scholar, Ramos-Mejia et al., 2012cRamos-Mejia V. Fraga M.F. Menendez P. iPSCs from cancer cells: challenges and opportunities.Trends Mol. Med. 2012; 18: 245-247Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, Yilmazer et al., 2015Yilmazer A. de Lazaro I. Taheri H. Reprogramming cancer cells: a novel approach for cancer therapy or a tool for disease-modeling?.Cancer Lett. 2015; 369: 1-8Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar), only a few reports have demonstrated successful reprogramming and, unfortunately, only seven of these studies reprogrammed human primary leukemias (the remaining studies used cell lines) (Bedel et al., 2013Bedel A. Pasquet J.M. Lippert E. Taillepierre M. Lagarde V. Dabernat S. Dubus P. Charaf L. Beliveau F. de Verneuil H. et al.Variable behavior of iPSCs derived from CML patients for response to TKI and hematopoietic differentiation.PLoS One. 2013; 8: e71596Crossref PubMed Scopus (20) Google Scholar, Carette et al., 2010Carette J.E. Pruszak J. Varadarajan M. Blomen V.A. Gokhale S. Camargo F.D. Wernig M. Jaenisch R. Brummelkamp T.R. Generation of iPSCs from cultured human malignant cells.Blood. 2010; 115: 4039-4042Crossref PubMed Scopus (186) Google Scholar, Gandre-Babbe et al., 2013Gandre-Babbe S. Paluru P. Aribeana C. Chou S.T. Bresolin S. Lu L. Sullivan S.K. Tasian S.K. Weng J. Favre H. et al.Patient-derived induced pluripotent stem cells recapitulate hematopoietic abnormalities of juvenile myelomonocytic leukemia.Blood. 2013; 121: 4925-4929Crossref PubMed Scopus (84) Google Scholar, Hu, 2014Hu K. All roads lead to induced pluripotent stem cells: the technologies of iPSC generation.Stem Cells Dev. 2014; 23: 1285-1300Crossref PubMed Scopus (67) Google Scholar, Kumano et al., 2012Kumano K. Arai S. Hosoi M. Taoka K. Takayama N. Otsu M. Nagae G. Ueda K. Nakazaki K. Kamikubo Y. et al.Generation of induced pluripotent stem cells from primary chronic myelogenous leukemia patient samples.Blood. 2012; 119: 6234-6242Crossref PubMed Scopus (123) Google Scholar, Yamamoto et al., 2015Yamamoto S. Otsu M. Matsuzaka E. Konishi C. Takagi H. Hanada S. Mochizuki S. Nakauchi H. Imai K. Tsuji K. Ebihara Y. Screening of drugs to treat 8p11 myeloproliferative syndrome using patient-derived induced pluripotent stem cells with fusion gene CEP110-FGFR1.PLoS One. 2015; 10: e0120841Google Scholar, Ye et al., 2009Ye Z. Zhan H. Mali P. Dowey S. Williams D.M. Jang Y.Y. Dang C.V. Spivak J.L. Moliterno A.R. Cheng L. Human-induced pluripotent stem cells from blood cells of healthy donors and patients with acquired blood disorders.Blood. 2009; 114: 5473-5480Crossref PubMed Scopus (313) Google Scholar) (Table S1). Intriguingly, iPSCs from hematological primary cancer cells have exclusively been generated from chronic leukemias of myeloid origin, including Philadelphia+ chronic myeloid leukemia (CML), primary myelofibrosis (PMF), JAK2-V617F+ polycythemia vera (PV), and juvenile myelomonocytic leukemia (JMML) (Table S1). iPSCs from acute myeloid leukemia (AML) or acute lymphoid leukemia (ALL) have not been reported so far, whereas iPSCs have been generated from normal myeloid and T cells (Bueno et al., 2016Bueno C. Sardina J.L. Di Stefano B. Romero-Moya D. Munoz-Lopez A. Ariza L. Chillon M.C. Balanzategui A. Castano J. Herreros A. et al.Reprogramming human B cells into induced pluripotent stem cells and its enhancement by C/EBPalpha.Leukemia. 2016; 30: 674-682Crossref PubMed Scopus (31) Google Scholar) and, very recently, from CD19+ B cells from human cord blood (CB), peripheral blood (PB), and fetal liver (FL) using non-integrative tetracistronic OCT4/KLF4/SOX2/MYC (OSKM)-expressing Sendai virus (SeV) (Bueno et al., 2016Bueno C. Sardina J.L. Di Stefano B. Romero-Moya D. Munoz-Lopez A. Ariza L. Chillon M.C. Balanzategui A. Castano J. Herreros A. et al.Reprogramming human B cells into induced pluripotent stem cells and its enhancement by C/EBPalpha.Leukemia. 2016; 30: 674-682Crossref PubMed Scopus (31) Google Scholar, Munoz-Lopez et al., 2016Munoz-Lopez A. van Roon E.H. Romero-Moya D. Lopez-Millan B. Stam R.W. Colomer D. Nakanishi M. Bueno C. Menendez P. Cellular ontogeny and hierarchy influence the reprogramming efficiency of human B cells into induced pluripotent stem cells.Stem Cells. 2016; 34: 581-587Crossref PubMed Scopus (13) Google Scholar). Here, we attempted to reprogram highly fluorescence-activated cell sorting (FACS)-purified (100% purity) leukemia blasts from three subtypes of B-ALL, t(4;11)/MLL-AF4+, t(1;11)+MLL-EPS15+, and t(12;21)/ETV6-RUNX1 B-ALL, to establish novel iPSC-based disease models to address the developmental impact of these leukemia-specific fusion genes on human stem cell fate. Our data demonstrate that despite multiple technical and biological reprogramming strategies, neither primary blasts nor B-ALL cell lines could be reprogrammed to pluripotency. Functional assays coupled with global transcriptome and DNA methylome profiling suggest a developmental/differentiation refractoriness of MLL-rearranged human B-ALL to reprogramming to pluripotency. iPSCs from primary leukemic cells harboring specific genetic mutations offer an unprecedented opportunity to understand how cancer-specific mutations impair tissue homeostasis by deregulating cell differentiation and proliferation. We attempted to reprogram blasts from t(4;11)/MLL-AF4+, t(1;11)+MLL-EPS15+, and t(12;21)/ETV6-RUNX1+ B-ALL. FACS-purified leukemic blasts (>99%, Figures 1A and 1B ) were infected (or transfected) with different combinations of monocistronic or polycistronic retroviral, lentiviral, and SeV vectors (or episomal vectors) expressing either OKSM or OKSL reprogramming factors (Table 1). No iPSC clones were generated when reprogramming factors were expressed via episomal vectors or viral retro-/lentivectors for any of the cytogenetically different leukemias tested (n = 7, Table 1). iPSC clones were exclusively generated when OKSM-expressing SeV vectors were employed (Figures 1C and 1D; Table 1). However, all of the resultant clones were negative for the corresponding fusion gene at the genomic (fluorescent in situ hybridization [FISH] and PCR) and RNA (RT-PCR) level (Figures 1E and 1F; Table 1).Table 1Summary of the Reprogramming Conditions Used in This Study and Their OutcomeReprogramming FactorsAdditional FactorsB-ALL t(4;11)B-ALL t(1;11)B-ALL t(12;21)Sort Purity (No. of Sorts)ClonesiFISH for MLL LocusPCR for MLL FusionLentiviral OKSM polycistronicNone√√√≥99% (single)0NANAascorbic acid√NDND≥99% (single)0NANAsodium butyrate√NDND≥99% (single)0NANAvalproic acid√NDND≥99% (single)0NANALiCl√NDND≥99% (single)0NANApLVX-mir302√√√≥99% (single)0NANAEpisomal OKSMnone√√√≥99% (single)0NANAascorbic acid√NDND≥99% (single)0NANAsodium butyrate√NDND≥99% (single)0NANAvalproic acid√NDND≥99% (single)0NANALiCl√NDND≥99% (single)0NANApLVX-mir302√√√≥99% (single)0NANAEpisomal OKSLnone√√√≥99% (single)0NANApLVX-mir302√√√≥99% (single)0NANARetroviral OKSMnone√√√≥99% (single)0NANASeV-OKSMnone√√√≥99% (single)yes∗negativenegativeSeVdp-OKSM polycistronicnone√√√≥99% (single)yes∗negativenegativesodium salicylate√√√≥99% (single)yes∗negativenegativedecitabine√NDND≥99% (single)yes∗negativenegativeiDot1L (SGC0946)√NDND≥99% (single)yes∗negativenegativeiDot1L (epz004777)√NDND≥99% (single)yes∗negativenegativeiMenin-MLL (iML2)√NDND≥99% (single)yes∗negativenegativeectopic c/EBPα√NDND≥99% (single)yes∗negativeNDiPTEN (bVp(HO)pic)√NDND≥99% (single)yes∗negativeNDc/EBPα + iPTEN√NDND≥99% (single)yes∗negativeNDdecitabine√NDND∼100% (double)0NANAtrichostatin A√NDND∼100% (double)0NANAvalproic acid√NDND∼100% (double)0NANAsodium butyrate√NDND∼100% (double)0NANAiSUV39H1 (Chaetocin)√NDND∼100% (double)0NANAiEZH2 (GSK126)√NDND∼100% (double)0NANAiEZH2 (DZNep)√NDND∼100% (double)0NANAiBRD4 (JQ1)√NDND∼100% (double)0NANAiCDK-P-TEFb (flavopiridol)√NDND∼100% (double)0NANAascorbic acid√NDND∼100% (double)0NANAoctyl-α-ketoglutarate√NDND∼100% (double)0NANAshRing1a MOI = 10√NDND∼100% (double)0NANAshMacroH2A MOI = 10√NDND∼100% (double)0NANAOKSM, Oct4, Klf4, Sox2, Myc; OKSL, Oct4, Klf4, Sox2, Lin28; SeVdp, Sendai vector-defective persistent; ND, not done; NA, not applicable; yes∗, the number of clones varies between 5 and 50. Open table in a new tab OKSM, Oct4, Klf4, Sox2, Myc; OKSL, Oct4, Klf4, Sox2, Lin28; SeVdp, Sendai vector-defective persistent; ND, not done; NA, not applicable; yes∗, the number of clones varies between 5 and 50. Several molecules that promote or enhance reprogramming, so-called reprogramming “boosters,” have been reported (Esteban et al., 2010Esteban M.A. Wang T. Qin B. Yang J. Qin D. Cai J. Li W. Weng Z. Chen J. Ni S. et al.Vitamin C enhances the generation of mouse and human induced pluripotent stem cells.Cell Stem Cell. 2010; 6: 71-79Abstract Full Text Full Text PDF PubMed Scopus (792) Google Scholar, Goyal et al., 2013Goyal A. Chavez S.L. Reijo Pera R.A. Generation of human induced pluripotent stem cells using epigenetic regulators reveals a germ cell-like identity in partially reprogrammed colonies.PLoS One. 2013; 8: e82838Crossref PubMed Scopus (9) Google Scholar, Onder et al., 2012Onder T.T. Kara N. Cherry A. Sinha A.U. Zhu N. Bernt K.M. Cahan P. Marcarci B.O. Unternaehrer J. Gupta P.B. et al.Chromatin-modifying enzymes as modulators of reprogramming.Nature. 2012; 483: 598-602Crossref PubMed Scopus (500) Google Scholar, Soria-Valles et al., 2015Soria-Valles C. Osorio F.G. Gutierrez-Fernandez A. De Los Angeles A. Bueno C. Menendez P. Martin-Subero J.I. Daley G.Q. Freije J.M. Lopez-Otin C. NF-kappaB activation impairs somatic cell reprogramming in ageing.Nat. Cell Biol. 2015; 17: 1004-1013Crossref PubMed Scopus (78) Google Scholar, Zhang and Wu, 2013Zhang Z. Wu W.S. Sodium butyrate promotes generation of human induced pluripotent stem cells through induction of the miR302/367 cluster.Stem Cells Dev. 2013; 22: 2268-2277Crossref PubMed Scopus (47) Google Scholar). SeV-OKSM-mediated reprogramming experiments were performed (mainly with MLL-AF4+ B-ALL blasts) using many reprogramming epigenetic/transcriptomic factors described to improve reprogramming (Hu, 2014Hu K. All roads lead to induced pluripotent stem cells: the technologies of iPSC generation.Stem Cells Dev. 2014; 23: 1285-1300Crossref PubMed Scopus (67) Google Scholar, Lin and Wu, 2015Lin T. Wu S. Reprogramming with small molecules instead of exogenous transcription factors.Stem Cells Int. 2015; 2015: 794632Crossref PubMed Scopus (56) Google Scholar). Although some of these factors enhanced the reprogramming efficiency (Figure 1G), FISH and RT-PCR assays revealed the absence of the fusion gene in resulting clones, indicating that residual non-leukemic myeloid cells were reprogrammed to pluripotency (Table 1). Furthermore, healthy adult B cells are known to be difficult to reprogram. We therefore attempted to reprogram MLL-AF4+ B cell blasts using SeV-OKSM in combination with (1) compounds which specifically target MLL fusion-driven signaling such as the Dot1L inhibitor and an inhibitor of Menin-MLL interaction (He et al., 2016He S. Malik B. Borkin D. Miao H. Shukla S. Kempinska K. Purohit T. Wang J. Chen L. Parkin B. et al.Menin-MLL inhibitors block oncogenic transformation by MLL-fusion proteins in a fusion partner-independent manner.Leukemia. 2016; 30: 508-513Crossref PubMed Scopus (37) Google Scholar) (2) the lymphoid “path breaker” cEBPα (Bueno et al., 2016Bueno C. Sardina J.L. Di Stefano B. Romero-Moya D. Munoz-Lopez A. Ariza L. Chillon M.C. Balanzategui A. Castano J. Herreros A. et al.Reprogramming human B cells into induced pluripotent stem cells and its enhancement by C/EBPalpha.Leukemia. 2016; 30: 674-682Crossref PubMed Scopus (31) Google Scholar, Di Stefano et al., 2014Di Stefano B. Sardina J.L. van Oevelen C. Collombet S. Kallin E.M. Vicent G.P. Lu J. Thieffry D. Beato M. Graf T. C/EBPalpha poises B cells for rapid reprogramming into induced pluripotent stem cells.Nature. 2014; 506: 235-239Crossref PubMed Scopus (154) Google Scholar), or (3) phosphatase and tensin homolog (PTEN) inhibitors that constitutively activate the phosphoinositol 3-kinase (PI3K) pathway resulting in increased iPSC generation (Liao et al., 2013Liao J. Marumoto T. Yamaguchi S. Okano S. Takeda N. Sakamoto C. Kawano H. Nii T. Miyamato S. Nagai Y. et al.Inhibition of PTEN tumor suppressor promotes the generation of induced pluripotent stem cells.Mol. Ther. 2013; 21: 1242-1250Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) and rescue of B cell receptor (BCR)-defective B cells (Srinivasan et al., 2009Srinivasan L. Sasaki Y. Calado D.P. Zhang B. Paik J.H. DePinho R.A. Kutok J.L. Kearney J.F. Otipoby K.L. Rajewsky K. PI3 kinase signals BCR-dependent mature B cell survival.Cell. 2009; 139: 573-586Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar). Although these conditions rendered iPSC clones, they were consistently negative for the MLL fusions (Table 1; Figures S1A and S1B). Importantly, OKSM-SeV-mediated reprogramming, with or without additional reprogramming boosters, of MLL-AF4+ leukemic blasts after double FACS sorting (virtually 100% purity) rendered no iPSC colonies, indicating that the limited number of iPSC clones lacking the MLL fusion were derived from easy-to-reprogram residual/contaminating non-leukemic myeloid cells (Table 1). Because human acute leukemias do not proliferate in vitro, we hypothesized that successful iPSC generation from leukemic blasts would rely on our ability to induce their proliferation. As a first approach, we used human leukemic B cell lines derived from B-ALL patients with MLL rearrangements (SEM and THP1) or with ETV6-RUNX1 (REH), rather than non-proliferating primary blasts. To this end, B cell lines were infected with OKSM-, OKSL-, or OKSML-expressing SeV vectors alone or in combination with epigenetic and transcriptomic reprogramming boosters; however, no iPSC clones could be generated (Figure S2A and Table 2). Moreover, immortalized B cell lines primed with decitabine and trichostatin A (TSA) prior to OKSM-SeV infection and then exposed to the aforementioned additional chemical inducers, also failed to generate iPSC clones (Figure S2B and Table 2). In another approach, SEM cells were stably knocked down for genes reported to act as barriers to induced pluripotency prior to OKSM-SeV infection (Gaspar-Maia et al., 2013Gaspar-Maia A. Qadeer Z.A. Hasson D. Ratnakumar K. Leu N.A. Leroy G. Liu S. Costanzi C. Valle-Garcia D. Schaniel C. et al.MacroH2A histone variants act as a barrier upon reprogramming towards pluripotency.Nat. Commun. 2013; 4: 1565Crossref PubMed Scopus (147) Google Scholar, Menendez et al., 2010Menendez S. Camus S. Izpisua Belmonte J.C. p53: guardian of reprogramming.Cell Cycle. 2010; 9: 3887-3891Crossref PubMed Scopus (61) Google Scholar, Nashun et al., 2015Nashun B. Hill P.W. Hajkova P. Reprogramming of cell fate: epigenetic memory and the erasure of memories past.EMBO J. 2015; 34: 1296-1308Crossref PubMed Scopus (111) Google Scholar, Pasque et al., 2011Pasque V. Gillich A. Garrett N. Gurdon J.B. Histone variant macroH2A confers resistance to nuclear reprogramming.EMBO J. 2011; 30: 2373-2387Crossref PubMed Scopus (115) Google Scholar). Intriguingly, small hairpin RNA (shRNA)-mediated knockdown of the tumor suppressor p53, the master B cell transcription factor Pax5, the Polycomb protein RING1a, and the histone variant macroH2A1 failed to facilitate the generation of iPSCs (Table 2; Figures S1C and S1D). Combination of p53 knockdown with 7 days’ treatment with demethylating agents (5-azacytidine, decitabine) before and after OKSM infection also failed to generate iPSCs. The knockdown of macroH2A1 was shown to reactivate a reporter gene on the inactive X chromosome only when combined with decitabine and TSA (Hernandez-Munoz et al., 2005Hernandez-Munoz I. Lund A.H. van der Stoop P. Boutsma E. Muijrers I. Verhoeven E. Nusinow D.A. Panning B. Marahrens Y. van Lohuizen M. Stable X chromosome inactivation involves the PRC1 Polycomb complex and requires histone MACROH2A1 and the CULLIN3/SPOP ubiquitin E3 ligase.Proc. Natl. Acad. Sci. USA. 2005; 102: 7635-7640Crossref PubMed Scopus (249) Google Scholar). As reactivation of the inactive X is a hallmark of reprogramming (Ohhata and Wutz, 2013Ohhata T. Wutz A. Reactivation of the inactive X chromosome in development and reprogramming.Cell Mol. Life Sci. 2013; 70: 2443-2461Crossref PubMed Scopus (56) Google Scholar), we tested the same and other triple combinations but found that SEM cells remained resistant to OKSM-induced reprogramming (Table 2).Table 2Summary of the Conditions Used to Reprogram the Leukemic B Cell Lines SEM, THP1, and REHReprogramming FactorsAdditional FactorsSEM t(4;11)THP1 t(9;11)REH t(12;21)ClonesiFISHGenomic PCRSeVdp-OSKM polycistronicnone√√√noNANAshp53 (MOI = 10)√√√noNANAshPax5 (MOI = 10)√√√noNANAcEBPα (MOI = 10)√√√noNANAShRING1a (MOI = 10)√NDNDnoNANAshMacroH2A1 (MOI = 10)√NDNDnoNANAiPTEN (bVp(HO)pic)√√√noNANAdecitabine√NDNDnoNANAiDot1L (SGC0946)√NDNDnoNANAiDot1L (epz004777)√NDNDnoNANAiMenin-Dot1L (iML2)√NDNDnoNANAtrichostatin A√NDNDnoNANAvalproic acid√NDNDnoNANAsodium butyrate√NDNDnoNANAsodium salicylate√NDNDnoNANAiSUV39H1 (Chaetocin)√NDNDnoNANAiEZH2 (GSK126)√NDNDnoNANAiEZH2 (DZNep)√NDNDnoNANAiBRD4 (JQ1)√NDNDnoNANAiCDK/iP-TEFb (Flavopiridol)√NDNDnoNANAascorbic acid√NDNDnoNANAoctyl-α-ketoglutarate√NDNDnoNANASeVdp-OSKL polycistronicnone√√√noNANASeVdp-OSKLN polycistronicnone√√√noNANASeVdp-OSKM polycistronic + decitabine 0.1 μM + trichostatin A 2 μMnone√√√noNANAshRING1a MOI = 10√NDNDnoNANAshMacroH2A1 MOI = 10√NDNDnoNANAiDot1L (SGC09469)√NDNDnoNANAiDot1L (epz004777)√NDNDnoNANAiMenin-MLL (iML2)√NDNDnoNANAvalproic acid√NDNDnoNANAsodium butyrate√NDNDnoNANAsodium salicylate√NDNDnoNANAiSUV39H1 (Chaetocin)√NDNDnoNANAiEZH2 (GSK126)√NDNDnoNANAiEZH2 (DZNep)√NDNDnoNANAiBRD4 (JQ1)√NDNDnoNANAiCDK/iP-TEFb (flavopiridol)√NDNDnoNANAascorbic acid√NDNDnoNANAoctyl-α-ketoglutarate√NDNDnoNANAND, not done; NA, not analyzed. Open table in a new tab ND, not done; NA, not analyzed. We next induced primary blasts to proliferate through xenograft expansion. Two approaches were followed: (1) in vivo expansion of OKSM-SeV-infected primary B cell blasts or (2) OKSM-SeV infection of in vivo expanded primary B cell blasts. In the second scenario, engrafted mice were treated with iDoT1L, decitabine, or left untreated, to (epi)-genetically prime the blasts prior to OKSM-SeV-infection (Figure 2A ). Although these strategies generated some iPSC clones after in vivo expansion of primary blasts in xenografted mice, all iPSCs analyzed lacked the MLL fusion gene by FISH and PCR, and were of mouse origin (Figures 2A and 2B). Together these results show that in vivo expanded leukemic blasts consistently failed to be reprogrammed. Our results show that neither primary MLL-AF4+ blasts nor proliferating leukemic B cell lines can be reprogrammed. However, and in line with previous work (Munoz-Lopez et al., 2016Munoz-Lopez A. van Roon E.H. Romero-Moya D. Lopez-Millan B. Stam R.W. Colomer D. Nakanishi M. Bueno C. Menendez P. Cellular ontogeny and hierarchy influence the reprogramming efficiency of human B cells into induced pluripotent stem cells.Stem Cells. 2016; 34: 581-587Crossref PubMed Scopus (13) Google Scholar), Epstein-Barr virus (EBV)-immortalized healthy B cells as well as healthy pro-B and pre-B cells could be successfully reprogrammed (Figure S2C), suggesting that the leukemia-initiating event (e.g., MLL fusion genes) may represent a reprogramming barrier. To test this idea, we lentivirally transduced both CB-CD34+ hematopoietic stem/progenitor cells (HSPCs) and CD34+CD19+ B cell progenitors with MLL-AF4-GFP, and after several days infected MLL-AF4-expressing CD34+ and CD34+CD19+ cells with OKSM-SeV (Bueno et al., 2015Bueno C. van Roon E.H. Muñoz-López A. Sanjuan-Pla A. Juan M. Navarro A. Stam R.W. Menendez P. Immunophenotypic analysis and quantification of B-1 and B-2 B cells during human fetal hematopoietic development.Leukemia. 2015; 30: 1603-1606Crossref PubMed Scopus (15) Google Scholar). MLL-AF4 expression did not impair the generation of iPSCs, and the reprogramming efficiency was similar to that of GFP-transduced CD34+ HSPCs (Figure 3A ) and CD34+CD19+ B cell progenitors (Figure S3A). Resulting iPSC clones displayed human embryonic stem cell (hESC)-like morphology and expressed MLL-AF4-GFP (Figures 3B and S3B). Further characterization revealed that MLL-AF4 was present in the majority of the iPSC clones and was always expressed (Figures 3C, 3D, and S3B) after ten passages. In addition, MLL-AF4-expressing iPSC clones were OKSM transgene independent (Figure 3E), diploid (Figure 3F), positive for alkaline phosphatase (Figure 3G), and expressed the pluripotency factors NANOG, OCT4, SOX2, REX1, DNMT3β, and CRIPTO (Figure 3H) and the surface markers TRA-1-60, SSEA3, and SSEA4 (Figure 3I). Importantly, iPSCs derived from MLL-AF4-expressing CD34+CD19+ B cell progenitors carried complete VDJH immunoglobulin gene monoclonal rearrangements, confirming the B lineage identity (Figure S3C). Collectively, these results suggest that MLL-AF4 expression does not seem to represent a reprogramming barrier in either CD34+ cells or CD34+CD19+ B cell progenitors, and is compatible with pluripotency. To identify patterns of gene expression that might provide a molecular explanation for the refractoriness of leukemic blasts to reprogramming, we compared gene expression profiles of FACS-purified MLL-AF4+ blasts from infant B-ALL (n = 3) with hematopoietic stem cells (HSCs) (n = 2), B cell hematopoietic progenitor cells (HPCs) (n = 2), and myeloid HPCs (n = 2) from healthy CB. A heatmap representation of hierarchical clustering of genes differentially expressed (2-fold regulated; p < 0.01) in MLL-AF4+ blasts versus healthy HSPCs is shown in Figure 4A . A total of 87 genes were differentially expressed in MLL-AF4+ blasts (Figures 4B and 4C). To gain insight into the biological functions affected by differentially expressed genes, we performed gene ontology (GO) analysis comparing MLL-AF4+ blasts with normal HSPCs (Figure 4D). Among the top significant GO biological processes enriched in MLL-AF4+ blasts, we found “cell differentiation,” “cell morphogenesis,” “developmental process,” and “cell proliferation” (Figure 4C), suggesting that the intrinsic developmental (differentiation) blockage and proliferative defects of leukemic blasts, rather than leukemia-specific genetic alterations, may constitute a reprogramming barrier. Similarly, to identify potential DNA methylation changes explaining the refractoriness of leukemic blasts to reprogramming, we performed global DNA methylation (LINE-1) profiling on FACS-purified MLL-AF4+ blasts from infant B-ALL (n = 3), B cell HPCs (n = 2), and MLL-AF4-expressing CD34+ HSPCs. Although no major quantitative changes in global DNA methylation were revealed by bisulfite pyrosequencing (Figure 5A ), DNA methylation 450K BeadChip arrays identified ∼6,700 CpG sites differentially methylated (dmCpGs; false discovery rate <0.05) between MLL-AF4+ blasts and both B cell HPCs and MLL-AF4-expressing HSPCs (Figures 5B and 5C). Specifically, 1,691 dmCpGs were hypomethylated and 5,012 CpGs hypermethylated in MLL-AF4+ leukemic blasts (Figure 5C). GO analysis of hypermethylated dmCpGs revealed “cell differentiation,” “cell morphogenesis,” and “developmental process” as significant biological processes enriched in MLL-AF4+ blasts (Figure 5D). GO analysis of hypomethylated dmCpGs identified RAS/JAK-STAT/MAPK activities (through which BCR-mediated signaling regulates B cell activation and differentiation) (Marshall et al., 2000Marshall A.J. Niiro H. Yun T.J. Clark E.A. Regulation of B-cell activation and differentiation by the phosphatidylinositol 3-kinase and phospholipase Cgamma pathway.Immunol. Rev. 2000; 176: 30-46Crossref PubMed Scopus (122) Google Scholar) as significant biological processes enriched in MLL-AF4+ blasts (Figure 5D). Thus, in line with the transcriptome data, these results suggest that the intrinsic differentiation blockage and proliferative status of leukemic blasts constitute a bona fide reprogramming barrier. iPSCs reprogrammed from cancer cells have the potential to illuminate molecular mechanisms underlying the pathogenesis of cancer (Barrett et al., 2014Barrett R. Ornelas L. Yeager N. Mandefro B. Sahabian A. Lenaeus L. Targan S.R. Svendsen C.N. Sareen D. Reliable generation of induced pluripotent stem cells from human lymphoblastoid cell lines.Stem Cells Transl. Med. 2014; 3: 1429-1434Crossref PubMed Scopus (54) Google Scholar, Curry et al., 2015Curry E.L. Moad M. Robson C.N. Heer R. Using induced pluripotent stem cells as a tool for modelling carcinogenesis.World J. Stem Cells. 2015; 7: 461-469Crossref PubMed Google Scholar, Ramos-Mejia et al., 2012cRamos-Mejia V. Fraga M.F. Menendez P. iPSCs from cancer cells: challenges and oppo" @default.
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• W2521164667 date "2016-10-01" @default.
• W2521164667 modified "2023-10-12" @default.
• W2521164667 title "Development Refractoriness of MLL-Rearranged Human B Cell Acute Leukemias to Reprogramming into Pluripotency" @default.
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• W2521164667 doi "https://doi.org/10.1016/j.stemcr.2016.08.013" @default.
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