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The reprogramming of human acute myeloid leukemia (AML) cells into induced pluripotent stem cell (iPSC) lines could provide new faithful genetic models of AML, but is currently hindered by low success rates and uncertainty about whether iPSC-derived cells resemble their primary counterparts. Here we developed a reprogramming method tailored to cancer cells, with which we generated iPSCs from 15 patients representing all major genetic groups of AML. These AML-iPSCs retain genetic fidelity and produce transplantable hematopoietic cells with hallmark phenotypic leukemic features. Critically, single-cell transcriptomics reveal that, upon xenotransplantation, iPSC-derived leukemias faithfully mimic the primary patient-matched xenografts. Transplantation of iPSC-derived leukemias capturing a clone and subclone from the same patient allowed us to isolate the contribution of a FLT3-ITD mutation to the AML phenotype. The results and resources reported here can transform basic and preclinical cancer research of AML and other human cancers. Significance: We report the generation of patient-derived iPSC models of all major genetic groups of human AML. These exhibit phenotypic hallmarks of AML in vitro and in vivo, inform the clonal hierarchy and clonal dynamics of human AML, and exhibit striking similarity to patient-matched primary leukemias upon xenotransplantation. See related commentary by Doulatov.

Supplemental Figure 1. Generation of a panel of iPSCs from patients with AML. Supplemental Figure 2. Reprogramming aids reconstruction of the evolutionary history and clonal composition of AML. Supplemental Figure 3. Transplantation of AML-iPSCs into immunodeficient mice. Supplemental Figure 4. Developmental block in a subset of AML-iPSC lines. Supplemental Figure 5. Transplantation of primary AML cells and patient-matched AMLiPSC lines. Supplemental Figure 6. Single-cell RNA-sequencing analyses of matched primary and iPSC-derived leukemia cells from patient AML-47. Supplemental Figure 7. Cell cycle and pseudotime analyses. Supplemental Figure 8. Comparison of scRNA-Seq data integration and clustering methods and pseudobulk differential gene expression analyses.

Table S1. Patient characteristics. AML: acute myeloid leukemia; MDS: myelodysplastic syndrome; MPN: myeloproliferative neoplasm; ET: essential thrombocythemia; PBMCs: peripheral blood mononuclear cells; BMMCs: bone marrow mononuclear cells; PDX: patient-derived xenografts Table S2. All patient samples used in this study with genetic characterization and reprogramming outcomes. Blue font denotes partially reprogrammed (as opposed to bona fide iPSC) colonies and clones. Table S3. All AML-iPSC lines phenotypically characterized. Table S4. Top 50 upregulated genes (highest log2 fold change) in each cluster. Table S5. Primers used for genotyping. Table S6. Primers used for qRT-PCR analyses.

The reprogramming of human acute myeloid leukemia (AML) cells into induced pluripotent stem cell (iPSC) lines could provide new faithful genetic models of AML, but is currently hindered by low success rates and uncertainty about whether iPSC-derived cells resemble their primary counterparts. Here we developed a reprogramming method tailored to cancer cells, with which we generated iPSCs from 15 patients representing all major genetic groups of AML. These AML-iPSCs retain genetic fidelity and produce transplantable hematopoietic cells with hallmark phenotypic leukemic features. Critically, single-cell transcriptomics reveal that, upon xenotransplantation, iPSC-derived leukemias faithfully mimic the primary patient-matched xenografts. Transplantation of iPSC-derived leukemias capturing a clone and subclone from the same patient allowed us to isolate the contribution of a FLT3-ITD mutation to the AML phenotype. The results and resources reported here can transform basic and preclinical cancer research of AML and other human cancers.Significance:We report the generation of patient-derived iPSC models of all major genetic groups of human AML. These exhibit phenotypic hallmarks of AML in vitro and in vivo, inform the clonal hierarchy and clonal dynamics of human AML, and exhibit striking similarity to patient-matched primary leukemias upon xenotransplantation.See related commentary by Doulatov.

Supplementary Data from Loss of PBRM1 Alters Promoter Histone Modifications and Activates ALDH1A1 to Drive Renal Cell Carcinoma

Supplementary Figure from Loss of PBRM1 Alters Promoter Histone Modifications and Activates ALDH1A1 to Drive Renal Cell Carcinoma

The reprogramming of malignant cells to pluripotency has presented considerable challenges, hindering the application of induced pluripotent stem cell (iPSC) modeling technology to human cancers. In addition, how well iPSC-derived cells resemble their primary counterparts is currently largely unknown. We developed a reprogramming method tailored to human malignancies, “Complete Capture of Mutational Burden” (CCoMB), that combines comprehensive genetic characterization of the starting sample and inference of its clonal architecture with large-scale screening of clonally reprogrammed colonies. Using this method we were able to generate a panel of iPSC lines representing all major genetic groups of acute myeloid leukemia (AML). Specifically, we derived iPSC lines from 15 patients representing all major genetic groups of AML - PML-RARA; chromatin-spliceosome; TP53-mutated/aneuploidy; AML1-ETO, MLL-rearranged; NPM1-mutated and others - collectively capturing 21 distinct genotypes and 24 driver genetic lesions (mutations, translocations, deletions). Matched normal iPSCs were derived from 7 of these patients. Reprogramming to iPSCs captured both preleukemic (CH/initiating mutation only) and fully leukemic clones (baring the full set of patient mutations). In almost all cases, reprogramming informed reconstruction of the evolutionary hierarchy of the AML, with unexpected hierarchies unveiled in 4 of the cases. These AML-iPSCs retain genetic fidelity and, upon in vitro hematopoietic differentiation, produce hematopoietic cells with hallmark phenotypic leukemic features, including serial engraftment of a lethal myeloid leukemia into immunocompromised mice and extended self-renewal in vitro. Transplantation of cells derived from iPSCs representing two distinct AML clones from each of 3 patients revealed that these mimic the clonal dynamics in the patients, with more advanced clones showing increased representation in the xenografts, compared to the earlier clones. To compare the iPSC-derived to the primary leukemias, we performed single-cell transcriptomics analyses in patient-matched iPSC-derived and primary leukemic cells from 3 patients, both ex vivo/in vitro and after transplantation into NSGS mice. Clustering analyses identified cell types corresponding to primitive hematopoietic stem cell (HSC)/multipotent progenitor (MPP), hematopoietic progenitor cells (HPCs) and more mature myelomonocytic lineage cells in all samples from all patients, at varying frequencies. Leukemias derived through in vitro differentiation from iPSC lines exhibited both similarities, as well as differences, in their cellular composition and transcriptome, compared to the patient-matched ex vivo leukemias. However, upon transplantation of the same leukemias into NSGS mice, iPSC-derived xenografts were strikingly similar to the patient-derived xenografts. iPSC-derived leukemic cells exhibited a more stem/progenitor cell phenotype in vitro, with progressive maturation along the myeloid lineage upon primary and, even more, upon secondary transplantation, mimicking primary xenografts. In summary, our results reveal very few true biological barriers to the reprogramming of AML cells and show that AML-iPSC-derived leukemias faithfully mimic the primary patient leukemias upon xenotransplantation. Citation Format: Andriana Kotini, Saul Carcamo, Nataly Cruz-Rodriguez, Malgorzata Olszewska, Dan Hasson, Eirini Papapetrou. Patient-derived iPSCs faithfully represent the genetic diversity and cellular architecture of human acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB363.

Background: Small cell lung cancer (SCLC) is an exceptionally aggressive disease with limited treatment options that typically result in transient responses. SCLC is responsible for approximately 250,000 deaths globally per year. Major hurdles to improving SCLC treatment include the development of rapid chemo-resistance and limited second-line therapies. Lurbinectedin is FDA approved as a second-line treatment for SCLC but shows a response in a subset of patients. Therefore, improved mechanistic understanding and identifying predictive biomarkers of lurbinectedin treatment is a major unmet clinical need. Methods: We treated SCLC cell lines and patient-derived xenograft (PDX) models from all major SCLC subtypes with lurbinectedin. Lurbinectedin-mediated changes in signaling pathways were studied by bulk RNA sequencing, western blot, and flow cytometry. Anti-tumor efficacy and toxicity studies were performed in vivo. Result: All human and PDX-derived SCLC cell lines showed sensitivity to lurbinectedin at a nano-molar concentration ranging from 1.905 to 30 nM. Bulk RNA-seq analysis showed lurbinectedin induced changes in neuroendocrine phenotype, DNA damage response and tumor progression markers in vitro. Single agent treatment of lurbinectedin showed remarkable anti-tumor efficacy in an ASCL1-driven PDX model. RNA sequencing analysis identified modulation of genes in multiple signaling pathways including PI3K-AKT, apoptosis and EMT to be significantly associated with the lurbinectedin response in PDX models. Conclusion: There is an immediate need to understand the subsets of SCLC that would be most sensitive to lurbinectedin and identify predictive biomarkers. We demonstrate MYC as a predictive biomarker for lurbinectedin response. We are the first to show that single agent lurbinectedin shows remarkable anti-tumor efficacy in a ASCL1-driven PDX models of SCLC. Furthermore, our pre and post-lurbinectedin treatment transcriptomic analysis identify the pathways that may contribute to primary or acquired resistance to lurbinectedin in SCLC. Finally, we identity candidate targets that would guide the design of future combination clinical trials with lurbinectedin in SCLC. Citation Format: Subhamoy Chakraborty, Charles Coleman, Deniz Demircioglu, Dan Hasson, Triparna Sen. Preclinical analysis identifies predictive biomarker and potential pathways of resistance to lurbinectedin treatment in small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB233.

Tumors exhibit widespread enhancer landscape reprogramming compared to normal tissue. The etiology is believed to be largely cell-intrinsic in non-hormonal cancers, attributed to such genomic alterations as focal amplification of non-coding regions, aberrant activation of transcription factors, and non-coding mutations creating de novo transcription factor binding sites. Here, using freshly resected primary CRC tumors and patient-matched adjacent normal colon epithelia, we find divergent epigenetic landscapes between primary CRC tumors and CRC cell lines. We identify a unique super-enhancer signature largely absent in cell culture. Intriguingly, this phenomenon extends to highly recurrent aberrant super-enhancers gained in CRC over patient-matched normal epithelium suggesting novel insight into the etiology of enhancer reprogramming in CRC and its downstream relevance to tumor biology. We find one such super-enhancer activated in epithelial cancer cells due to surrounding inflammation in the tumor microenvironment. CRISPR-dcas9-KRAB interference of this super-enhancer identifies PDZK1IP1 as its target gene. PDZK1IP1 is previously observed to be highly up-regulated in CRC. However, the mechanism behind its transcriptional activation is not fully understood. We restore both the super-enhancer and PDZK1IP1 levels following treatment with cytokines or xenotransplantation into nude mice, thus demonstrating its etiology via local tumor microenvironment acquisition. Deletion of inflammatory transcription factors RELA and STAT3 in human CRC cells inhibits PDZK1IP1 induction in xenografts. PDZK1IP1 appears to be critical for CRC growth in the setting of its super-enhancer induction as xenografts, but not in cell culture where the super-enhancer is absent and expression is largely silent. Building on its known role in glucose uptake via SGLT receptors, we demonstrate mechanistically that PDZK1IP1 enhances the reductive capacity CRC cancer cells via the pentose phosphate pathway using polar metabolomic profiling. We show this activation enables efficient growth under oxidative conditions both in vitro and in vivo, challenging the previous notion that PDZK1IP1 acts as a tumor suppressor in CRC. Collectively, these observations highlight the biologic significance of epigenomic profiling on patient-matched primary specimens and identify this microenvironment-acquired super-enhancer as an oncogenic driver in the setting of the inflamed tumor. Citation Format: Royce Zhou, Jia Xu, Tiphaine Martin, Alexis Zachem, John He, Sait Ozturk, Deniz Demircioglu, Ankita Bansal, Andrew Trotta, Bruno Giotti, Berkley Gryder, Yao Shen, Saul Carcamo, Xuewei Wu, Kaitlyn Bosch, Benjamin Hopkins, Alexander Tsankov, Randolph Steinhagen, Drew Jones, John Asara, Jerry Chipuk, Rachel Brody, Steven Itzkowitz, Iok In Christine Chio, Dan Hasson, Emily Bernstein, Ramon Parsons. A local tumor microenvironment acquired super-enhancer induces an oncogenic driver in colorectal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3481.

The process of cellular senescence generates a repressive chromatin environment, however, the role of histone tail modifications, histone variants and histone proteolytic cleavage in senescence remains unclear. Using well-characterized senescence models in human primary lung fibroblasts and melanocytes, we are investigating how chromatin alterations contribute to the senescence phenotype. Here we report novel histone H3 tail cleavage events in oncogene-induced and replicative senescence models mediated by the protease Cathepsin L. We find that cleaved forms of H3 are nucleosomal and that H3.3 is the preferred cleaved form of H3 - an H3 histone variant that is deposited independent of DNA replication. Ectopic expression of H3.3 and its cleavage product (H3.3cs1), which lacks the first twenty-one amino acids of the H3 tail, is sufficient to induce senescence. Furthermore, we find that chromatin incorporation of H3.3cs1 is mediated by the HUCA (HIRA/UBN1/CABIN1/ASF1a) histone chaperone complex. Genome-wide transcriptional profiling revealed that H3.3cs1 facilitates transcriptional silencing of cell cycle regulators including RB/E2F target genes, likely via the permanent removal of H3K4me3. Collectively, our studies have identified histone H3.3 and its proteolytically processed forms as key regulators of cellular senescence. Citation Format: Luis F. Duarte, Andrew R. J. Young, Zichen Wang, Hsan-Au Wu, Taniya Panda, Yan Kou, Avnish Kapoor, Dan Hasson, Nicholas R. Mills, Avi Ma'ayan, Masashi Narita, Emily Bernstein. Histone tail alterations in cellular senescence. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr SY10-04. doi:10.1158/1538-7445.AM2015-SY10-04

Malignant melanoma is the most lethal form of skin cancer with rising incidence. Once metastasis occurs, patients have a dismal prognosis, largely due to limited systemic treatment with chemotherapy and resistance to targeted therapies. Thus, effective therapies with long-term responses are currently lacking. Although much effort has focused on characterizing and targeting the genetic alterations in melanoma, the identification of epigenetic players remains poorly understood. Chromatin dynamics have recently been shown to exert a critical function in a number of cancers, including melanoma, and emerging evidence points towards a role of histone variants as key regulatory molecules in cancer. H2A.Z is a highly conserved H2A variant, harboring two different isoforms in vertebrates, H2A.Z.1 and H2A.Z.2. High levels of H2A.Z promote cell proliferation in breast, prostate and bladder cancers, however studies so far have focused primarily on H2A.Z.1 or did not clearly distinguish between the two isoforms. Here, we report a role for the unappreciated isoform H2A.Z.2 as a mediator of cell proliferation and drug sensitivity in malignant melanoma. To our knowledge, this is the first evidence to implicate a distinct role for this H2A.Z isoform in any tumor type. While both H2A.Z.1 and H2A.Z.2 are highly expressed in metastatic melanoma and correlate with decreased patient survival, only H2A.Z.2 deficiency results in impaired cellular proliferation, which occurs through a G1 to S arrest. Integrated gene expression and ChIP-seq analyses revealed that H2A.Z.2 positively regulates E2F target genes, which are highly expressed and acquire a distinct H2A.Z occupancy signature over the promoter and gene body in metastatic cells. We further identified the BET (bromodomain and extraterminal domain) family member BRD2 as an H2A.Z-interacting protein in melanoma cells, and our data suggest that H2A.Z.2 exerts its oncogenic function by maintaining the global levels of BRD2 and histone H4 acetylation. Furthermore, H2A.Z.2 depletion sensitizes melanoma cells to targeted therapies and chemotherapy. Collectively, our findings implicate H2A.Z.2 as a driver of melanoma pathogenesis. Owing to the fact that histone modification is a reversible process, H2A.Z.2 and BRD2 hold translational potential for novel therapeutic strategies. Citation Format: Chiara Vardabasso, Alexandre Gaspar-Maia, Sebastian Punzeler, David Valle-Garcia, Dan Hasson, Tobias Straub, Eva C. Keilhauer, Thomas Strub, Taniya Panda, Miguel F. Segura, Chi-Yeh Chung, Amit K. Verma, Matthias Mann, Eva Hernando, Sandra B. Hake, Emily Bernstein. Histone variant H2A.Z.2 mediates proliferation and drug sensitivity of malignant melanoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr A12.

Oncogene-induced senescence (OIS) is characterized by irreversible loss of proliferative capacity. It has been proposed that OIS functions as a tumor suppressor mechanism, and several studies have demonstrated induction of senescence markers in pre-malignant lesions. In addition to profound morphological changes, significant alterations in chromatin structure occur during senescence including formation of senescence-associated heterochromatin foci (SAHF). We have used well-characterized senescence models in human primary lung fibroblasts and melanocytes to study how chromatin alterations contribute to the senescence phenotype. We discovered that the N'-terminal tail of histone H3 is proteolytically cleaved by the lysosomal protease Cathepsin L1 (CTSL1). We show that CTSL1 is induced during senescence both at the mRNA and protein levels, and that it is present in the chromatin fraction of senescent cells. Chemical and shRNA-mediated inhibition of CTSL1 activity effectively block H3 tail cleavage and leads to a SAHF formation defect. We identified two distinct cleaved forms of H3 in senescent cells, which are associated with chromatin and incorporated into nucleosomes. Using antibodies directed against specific post-translational modifications (PTMs) of the histone H3 tail, we have mapped the approximate locations of the cleavage sites. Furthermore, overexpression of flag-tagged cleaved versions of H3, but not the full-length histone, trigger robust senescence in IMR90 cells accompanied by a modest induction of SAHFs. Interestingly, we also detect cleaved H3 in chromatin extracted from benign nevi tissue samples, providing evidence of H3 tail cleavage in senescence models in vivo. Collectively, our results suggest that H3 tail cleavage may contribute significantly to the senescence phenotype, potentially through transcriptional regulation of key senescence target genes. This area of investigation is currently ongoing. Citation Format: Luis F. Duarte, Andrew R. Young, Hsan-Au Wu, Taniya Panda, Zichen Wang, Alexandre G. Maia, Dan Hasson, Avnish Kapoor, Masashi Narita, Emily Bernstein. Histone H3 cleavage via Cathepsin L drives a cellular senescence program. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2247. doi:10.1158/1538-7445.AM2014-2247