Chimeric Transcription Factor Research Articles

Single-cell multiomics profiling reveals heterogeneous transcriptional programs and microenvironment in DSRCTs

Desmoplastic small round cell tumor (DSRCT) is a rare, aggressive sarcoma driven by the EWSR1::WT1 chimeric transcription factor. Despite this unique oncogenic driver, DSRCT displays a polyphenotypic differentiation of unknown causality. Using single-cell multi-omics on 12 samples from five patients, we find that DSRCT tumor cells cluster into consistent subpopulations with partially overlapping lineage- and metabolism-related transcriptional programs. Invitro modeling shows that high EWSR1::WT1 DNA-binding activity associates with most lineage-related states, in contrast to glycolytic and profibrotic states. Single-cell chromatin accessibility analysis suggests that EWSR1::WT1 binding site variability may drive distinct lineage-related transcriptional programs, supporting some level of cell-intrinsic plasticity. Spatial transcriptomics reveals that glycolytic and profibrotic states specifically localize within hypoxic niches at the periphery of tumor cell islets, suggesting an additional role of tumor cell-extrinsic microenvironmental cues. We finally identify a single-cell transcriptomics-derived epithelial signature associated with improved patient survival, highlighting the clinical relevance of our findings.

Abstract 138: UCHL5 and PAX3-FOXO1 resolve replication stress in rhabdomyosarcoma

Abstract PAX3-FOXO1 positive rhabdomyosarcoma (RMS) is an aggressive pediatric sarcoma with a terrible prognosis. The resultant fusion acts as a chimeric transcription factor, in part remodeling chromatin to inhibit terminal differentiation and maintain cancer cell proliferation. As this oncoprotein is not directly druggable, we have used genetic screens to identify dependencies downstream of PAX3-FOXO1 to improve our understanding of pathogenesis and develop new therapeutic targets. We performed pooled CRISPRi screening in an isogenic system of a patient-derived RMS cell line with or without knockdown of PAX3-FOXO1. Loss of the ubiquitin hydroxylase UCHL5 led to selective depletion of PAX3-FOXO1 positive Rh30 cells compared to PAX3-FOXO1 knockdown cells, suggesting that the fusion creates UCHL5 dependence. We validated this finding in competition growth assays in additional fusion positive (FP) and fusion negative (FN) RMS cell lines, and observed UCHL5 dependency only in the former. By contrast, UCHL5 was dispensable for cell growth and survival in FN RMS cell lines. These data were recapitulated in the large DepMap dataset, which also identified the top genetic co-dependencies of UCHL5 as INO80-complex (INO80c) members. We therefore investigated whether the fusion selective effects of UCHL5 were mediated by the INO80c, of which UCHL5 is an N-terminal member. We observed that knockdown of another N-terminal component of INO80c, NFRKB, phenocopies the fusion-selective effects of UCHL5 loss. Conversely, the catalytic ATPase INO80 was pan-essential, suggesting that only N-terminal subunits of INO80c regulate PAX3-FOXO1 specific functions. INO80c plays an essential role in mitigating DNA replication stress. Indeed, loss of UCHL5 led to DNA damage as measured by γH2AX and slowing of replication forks as measured by fiber assays in FP, but not FN RMS. Unexpectedly, acute degradation of PAX3-FOXO1 also slowed DNA replication fork speed, suggesting a genetic model in which UCHL5 and PAX3-FOXO1 cooperate to resolve replication stress in FP RMS. Finally, in vivo experiments using cell line xenografts confirms that loss of UCHL5 slows the growth of FP RMS, but not FN RMS. These findings suggest that loss of UCHL5 induces replication stress in a fusion-specific manner, and offers both an opportunity to study the mechanisms by which the PAX3-FOXO1 oncoprotein mitigates replication stress, and to develop UCHL5 inhibitors as precision therapies for FP RMS patients. Citation Format: Amit J. Sabnis, Pushpendra K. Sahu, Yue Pan. UCHL5 and PAX3-FOXO1 resolve replication stress in rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 138.

Abstract 1081: A role for the oncogenic driver fusion protein EWS-FLI1 in the targeting of DR5 by INBRX-109 in Ewing sarcoma

Abstract Background: Tumor necrosis factor receptor superfamily member 10b (TNFRSF10b, also known as DR5) agonists are attractive targeted agents for cancer cells because these agents activate cellular apoptosis. Ewing sarcoma (ES) is a bone and soft-tissue sarcoma occurring in adolescence defined by a t(11;22)(q24;q12) balanced chromosomal translocation that results in chimeric transcription factor and oncogenic driver protein, EWS-FLI1. Independent studies and unbiased cell line screens have reported ES to be sensitive to DR5 targeting. INBRX-109 is a tetravalent, DR5-targeted antibody. Here, we analyzed the sensitivity of ES models to DR5 targeting by INBRX-109 and explored how the EWS-FLI1 transcriptional program impacts the continuum of sensitivity-resistance to this targeting. Methods: In a panel of 11 ES lines, in vitro sensitivity was assessed to both single agent and INBRX-109 combinations. A screen of 1,363 FDA approved compounds identified cooperative combinations that were prioritized mechanistically. Differential sensitivity for single agent and in combination with irinotecan was confirmed in vivo in xenograft models. Resistant clones were isolated from in vivo experiments and evaluated for DR5 expression and sensitivity. Cellular factors associated with sensitivity and resistance were determined by siRNA knockdown and Cleavage Under Targets and Tagmentation assay (CUT and Tag). Results: ES models showed variable sensitivity to INBRX-109, with a GI90 as low as 10 picomolar in some models. All models showed robust expression of DR5, but absolute expression of DR5 did not correlate with sensitivity. In vitro sensitivity was replicated in vivo, with all 7 mice bearing RD-ES xenografts demonstrating initial complete tumor regression and 3 of 7 mice demonstrating no recurrence beyond 90 days. Importantly, cells isolated from recurrent tumors showed a novel mechanism of resistance: direct repression of DR5 by EWS-FLI1. INBRX-109 was ineffective as a single agent at 10 nM in resistant ES models. However, there was striking synergy in vitro with SN38, the active metabolite of irinotecan, and this effect translated into tumor regressions of a resistant TC32 xenograft in vivo. The FDA compound screen captured novel synergy with a panel of agents. Conclusion: INBRX-109 is an exciting candidate for ES as a single agent in subsets of tumors or in combination with irinotecan, which is currently being evaluated in the clinic. Investigation of the exact relationship between EWS-FLI1 and sensitivity to DR5 targeting is ongoing. Initial findings suggest EWS-FLI1, combined with other disease modifying mutations, is a key mediator of this sensitivity. Further, EWS-FLI1 plays a direct role repressing DR5 in the setting of therapeutic challenge in one model in vivo. These findings highlight the potential for INBRX-109 in combination with irinotecan or novel candidates as therapies for ES patients. Citation Format: Curtis C. Parker, Lauren M. Gaetano, Elissa A. Boguslawski, Seneca Kinn-Gurzo, Rebecca Kaufman, Matthew C. Stout, Chase Deveraux, Patrick J. Grohar. A role for the oncogenic driver fusion protein EWS-FLI1 in the targeting of DR5 by INBRX-109 in Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1081.

Abstract 143: Molecular profiling of tumor-initiation events in rhabdomyosarcoma uncovers mitochondrial vulnerabilities

Abstract Background: Alveolar Rhabdomyosarcoma (ARMS) is a very aggressive soft tissue tumor affecting children and adolescents. It is found that pediatric patients with RMS harboring chimeric PAX fusion transcription factors (TFs) exhibit a greater incidence of tumor relapse, metastasis, and poor survival outcome, thereby underscoring the urgent need to develop effective therapies to treat this subtype of childhood cancer. Methods: We generated a novel ARMS model by stable transduction of myogenic progenitor cells with a cassette encoding the exact PAX3/7-FOXO1 fusion protein found in ARMS tumors. We analyzed genome-wide expression levels with RNA-seq and utilized Cut&Run to map the binding sites of PAX3/7-FOXO1, active histone marks, enhancers, and super-enhancers in our novel model. Hi-C and promoter capture HiC (pCHiC) were also performed to generate 3D chromatin conformation maps in cells expressing the oncogenic TFs. We compared these data with analogous datasets from orthotopic patient-derived xenografts (O-PDXs) of PAX3/7-FOXO1 fusion-positive ARMS tumors from St. Jude and established ARMS cell lines to validate whether our model system recapitulates ARMS tumors. CRISPRi was performed to silence key targets in two representative ARMS cell lines and proliferation was scored after silencing. Next, we found that ARMS tumors displayed anomalies in mitochondrial mass and numbers, metabolic parameters, and membrane potential by performing TEM, Seahorse assays, and FACS-based TMRM and mitotracker Red dye uptake in a panel of ARMS cells and PDX samples. We performed cell viability and colony forming assays in ARMS cells treated with single drugs and combinations of drugs targeting FGFR4 and mitochondrial metabolism. Results: Our data provide evidence to support muscle progenitors as potential cells-of-origin for ARMS. We showed that rewiring of chromatin architecture mediated by PAX3/7-FOXO1 TFs is evident across established cell lines, primary tumors as well as O-PDXs. By integrating PAX3/7-FOXO1 binding, RNA-seq, and pCHiC data, we identified key promoter-enhancer (P-En) interactions that are erased or established de novo at multiple loci of master TFs like MYOD, MYOG and MYCN. We also identified key regulators of proliferation, including FGFR4, and mitochondrial function that are essential for tumor growth and invasiveness. Our data indicated aberrant and enhanced mitochondrial metabolism in ARMS as well as selective sensitivity to mitochondrial inhibitors in RMS cells. Administration of these inhibitors preferentially inhibited cell viability and clonogenicity in ARMS, and prompted a pre-clinical phase II study to test these inhibitors at St. Jude Research Hospital. Conclusion: We present a novel treatment strategy leveraging the mitochondrial dependencies of fusion positive PAX-driven tumors which can be exploited for precision therapy in high-risk ARMS. Citation Format: Bhargab Kalita, Gerard Martinez-Cebrian, Justina McEvoy, Brittney Gordon, Kaley Blankenship, Asa Karlstorm, Elizabeth Stewart, Michael Dyer, Brian Dynlacht. Molecular profiling of tumor-initiation events in rhabdomyosarcoma uncovers mitochondrial vulnerabilities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 143.

Abstract 2861: Rhabdomyosarcoma fusion-oncogene alters the chromatin landscape to initially drive a neural signature in vivo

Abstract Fusion-positive rhabdomyosarcoma is an aggressive pediatric cancer lacking curative therapies, and outcomes for children with this disease have not improved in decades. The chimeric transcription factor PAX3::FOXO1 is the most common and lethal driver of fusion-positive rhabdomyosarcoma, and it consists of the DNA binding domains of PAX3 fused to the transactivation domain of FOXO1. Despite its well-established essentiality for tumorigenesis, how PAX3::FOXO1 initially generates a tumorigenic cell state has been challenging to study due to the lack of in vivo models expressing the fusion-oncogene at accessible early developmental time points. Here, we developed a novel zebrafish mRNA injection model that ubiquitously expresses PAX3::FOXO1 during gastrulation representing all three germ layers. Using this high-throughput model, we used ChIP-seq to identify initial in vivo PAX3::FOXO1 binding sites and targets. PAX3::FOXO1 has pioneering activity, where it can utilize partial-motif recognition to bind to inaccessible chromatin and alter chromatin structure. With biochemical and 2D chromatin sequencing approaches in our model, we find evidence for in vivo PAX3::FOXO1 pioneering activity. PAX3::FOXO1 consists of two DNA binding domains, a paired and homeobox domain, and we demonstrate a new mode of pioneering activity driven by its homeobox domain given a strong enrichment of homeobox-related motifs at PAX3::FOXO1 binding sites. PAX3::FOXO1 activity in developing zebrafish embryos results in an arrested development phenotype and transcriptional signatures enriched for pathways such as an inhibition of segmentation and myogenic development. Critically, we find that PAX3::FOXO1 directly activates neural-related gene targets by increasing chromatin accessibility and re-distribution of the active histone mark H3K27Ac to these loci. These discoveries are particularly striking because rhabdomyosarcoma is traditionally associated with skeletal muscle characteristics. With our findings, we hypothesize neural gene activation is a critical mechanism for fusion-positive rhabdomyosarcoma tumor initiation and could be a critical pathway across various stages of rhabdomyosarcoma tumorigenesis. This hypothesis agrees with our previous discovery of a novel PAX3::FOXO1 cooperating neural transcription factor, HES3, which we find is directly activated by PAX3::FOXO1 in our mRNA injection model. Our long-term goal is to utilize this versatile model to functionally evaluate cooperation between PAX3::FOXO1 and both canonical rhabdomyosarcoma and neural transcription factors. Our approach will employ a cross-species comparative analysis across this injection model, our zebrafish tumor models, other model systems, and patient data. This work will identify conserved targets and mechanisms of PAX3::FOXO1 tumorigenesis that could become new therapeutic vulnerabilities. Citation Format: Jack Kucinski, Cenny Taslim, Alexi Tallan, Matthew Cannon, Katherine Silvius, Benjamin Stanton, Genevieve Kendall. Rhabdomyosarcoma fusion-oncogene alters the chromatin landscape to initially drive a neural signature in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2861.

Abstract 2851: Characterization of neogenes in desmoplastic small round cell tumors

Abstract Desmoplastic Small Round Cell Tumor (DSRCT) is a rare, soft tissue sarcoma caused by the EWS::WT1 fusion protein (FP). The FP acts as an aberrant transcription factor with oncogenic properties. Recently, oncogenic chimeric transcription factors (OCTF), such as EWS::WT1, were discovered to induce the expression of novel transcripts in otherwise silent genomic regions. These neogenes (NGs) are highly specific to each OCTF and not detected in normal tissue. 37 NGs were found to be associated with the EWS::WT1 FP.We explored the NGs to characterize their expression and regulation in a cohort of MD Anderson Cancer Center patients. We annotated and counted the NGs from the bulk RNA-seq data of 37 specimens. After fusion detection, only 22/37 specimens harbored the EWS::WT1 FP. We found that only the same 22 specimens robustly expressed DSRCT-specific NGs. Interestingly the NGs were heterogeneously expressed, suggesting that there may be patient-specific differences. Single-nucleus RNA-sequencing (snRNA-seq) of Ewing sarcoma (ES), CIC::DUX4 sarcoma (CDS), DSRCT, and osteosarcoma revealed that only the three sarcomas driven by an OCTF robustly expressed NGs. Notably, the DSRCT-specific NGs were only expressed in DSRCT. Similarly, ES and CDS only expressed their subtype-specific NGs. Given the robust expression of the DSRCT-specific NGs in DSRCT, we asked if the EWS::WT1 FP regulated the expression of the NGs. Knockdown (KD) of the EWS::WT1 FP in four DSRCT cell lines showed a consistent reduction in DSRCT-specific NGs. While there was variability in NG expression and EWS::WT1 FP KD sensitivity, we identified seven NGs decreased by at least 75% in all four cell lines and an additional eight NGs with 75% reduction across three cell lines. BOD-DSRCT and SK-DSRCT2 had the largest number of NGs reduced after KD, with 25/37 and 20/37 NGs with 75% expression reduction, respectively. EWS::WT1 FP has two known isoforms generated by alternative splicing in the WT1 domain that includes or excludes three amino acids, lysine, threonine, and serine (KTS) between zinc fingers 3 and 4, producing E+KTS and E-KTS isoforms. By transducing one or both isoforms into the mesothelial cell line LP9, we found 3/37 NGs were induced by the E+KTS isoform and 17/37 were induced by the E-KTS isoform, suggesting that the E-KTS isoform is primarily responsible for NG expression. Current work is underway investigating the relationship between EWS::WT1 FP binding and NG expression and the potential for NG to produce novel peptides that could serve as DSRCT immunotherapy targets. Citation Format: Danh Truong, Justin Magrath, Sean Lee, Joseph Ludwig. Characterization of neogenes in desmoplastic small round cell tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2851.

Abstract 4739: Targeting resistance to BET inhibitors in Fusion-Positive Rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most frequent pediatric soft tissue sarcoma. Fusion-positive (FP-)RMS expressing the oncogenic chimeric transcription factor (TF) PAX3-FOXO1 (P3F) is at high risk of recurrence. FP-RMS cells show survival dependency on P3F. Despite P3F, as a TF, is considered undruggable, we and others demonstrated that P3F levels/functions can be epigenetically modulated. The BET protein and epigenetic reader BRD4 binds super-enhancers (SEs) to foster oncogenic transcription in cancer. We showed that P3F rewires the RMS enhancer landscape by recruiting BRD4 on oncogenic SEs. Accordingly, JQ1, a BET inhibitor (BETi) shuts down P3F functions halting tumor growth in vitro and in vivo. Therefore, BRD4 inhibition in FP-RMS results in a tumor subtype-specific vulnerability. Nonetheless, as for other targeted therapies, cancer cells can acquire resistance to BETi, thus, we investigated whether FP-RMS cells could develop BETi resistance. Therefore, we established resistant cells from two sensitive FP-RMS cell lines chronically exposed to escalating doses of JQ1, as a model of naturally acquired resistance. Our data show that the two established FP-RMS cell lines, acquired resistance by increasing JQ1 IC50 over the sensitive cells both in 2D and 3D settings. Compared to the sensitive ones, both the resistant cell lines show cross-resistance to other monovalent and bivalent BETi and degraders. Resistant cells also maintain the ability to form colonies in a clonogenic assay and to invade in a 3D Matrigel assay in the presence of JQ1. Moreover, conversely to sensitive cells, JQ1 treatment is unable to down-regulate P3F, MYCN and BCL2 (P3F target) levels in resistant cells. Furthermore, integration of transcriptomic and proteomic data reveals differentially modulated subset of genes/proteins among which MYCN and P3F targets, suggesting a restored activity of the TFs circuitry in resistant cells. Accordingly, MYCN silencing completely halts growth of resistant cells. In line, MYCN protein levels are reduced by JQ1 in a dose-dependent manner in sensitive but not in resistant cells and its half-life is 3 folds longer in resistant than in sensitive cells. Being MYCN protein stability affected by specific post-translational modifications among which the phosphorylation at Ser62 by ERKs, we evaluated the expression of phosphorylated MYCN and the activated form of ERK (pERK) and found both of them increased in resistant cells. In agreement, resistant cells are more sensitive to MEK-ERKs pathway inhibition, showing a lower IC50 of Trametinib (MEKi) compared to sensitive cells. Moreover, Trametinib and JQ1 co-treatment downregulates MYCN protein levels in resistant but not in sensitive cells, suggesting that ERKs overactivation could participate to BETi acquired resistance by stabilizing MYCN, thus representing an acquired vulnerability. The study has received funding from Ministero della Salute to SP. Citation Format: Matteo Cassandri, Erika Ferraro, Lucrezia D'Archivio, Francesca Aiello, Hsien-Chao Chou, Young K. Song, Berkley E. Gryder, Jun S. Wei, Simone Sidoli, Robert G. Hawley, Rossella Rota, Franco Locatelli, Javed Khan, Silvia Pomella. Targeting resistance to BET inhibitors in Fusion-Positive Rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4739.

Deciphering Acute Myeloid Leukemia Associated Transcription Factors in Human Primary CD34+ Hematopoietic Stem/Progenitor Cells.

Hemato-oncological diseases account for nearly 10% of all malignancies and can be classified into leukemia, lymphoma, myeloproliferative diseases, and myelodysplastic syndromes. The causes and prognosis of these disease entities are highly variable. Most entities are not permanently controllable and ultimately lead to the patient's death. At the molecular level, recurrent mutations including chromosomal translocations initiate the transformation from normal stem-/progenitor cells into malignant blasts finally floating the patient's bone marrow and blood system. In acute myeloid leukemia (AML), the so-called master transcription factors such as RUNX1, KMT2A, and HOX are frequently disrupted by chromosomal translocations, resulting in neomorphic oncogenic fusion genes. Triggering ex vivo expansion of primary human CD34+ stem/progenitor cells represents a distinct characteristic of such chimeric AML transcription factors. Regarding oncogenic mechanisms of AML, most studies focus on murine models. However, due to biological differences between mice and humans, findings are only partly transferable. This review focuses on the genetic manipulation of human CD34+ primary hematopoietic stem/progenitor cells derived from healthy donors to model acute myeloid leukemia cell growth. Analysis of defined single- or multi-hit human cellular AML models will elucidate molecular mechanisms of the development, maintenance, and potential molecular intervention strategies to counteract malignant human AML blast cell growth.

Development of new binary expression systems for plant synthetic biology.

A novel plant binary expression system was developed from the compactin biosynthetic pathway 27 of Penicillium citrinum ML-236B. The system achieved >fivefold activation of gene expression in 28 transgenic tobacco. A diverse and well-characterized genetic toolset is fundamental to achieve the overall goals of plant synthetic biology. To properly coordinate expression of a multigene pathway, this toolset should include binary systems that control gene expression at the level of transcription. In plants, few highly functional, orthogonal transcriptional regulators have been identified. Here, we describe the process of developing synthetic plant transcription factors using regulatory elements from the Penicillium citrinum ML-236B (compactin) pathway. This pathway contains several genes including mlcA and mlcC that are transcriptionally regulated in a dose-dependent manner by the activator mlcR. In Nicotiana benthamiana, we first expressed mlcR with several cognate synthetic promoters driving expression of GFP. Synthetic promoters contained operator sequences from the compactin gene cluster. Following identification of the most active synthetic promoter, the DNA-binding domain from mlcR was used to generate chimeric transcription factors containing variable activation domains, including QF from the Neurospora crassa Q-system. Activity was measured at both protein and RNA levels which correlated with an R2 value of 0.94. A synthetic transcription factor with a QF activation domain increased gene expression from its synthetic promoter up to sixfold in N. benthamiana. Two systems were characterized in transgenic tobacco plants. The QF-based plants maintained high expression in tobacco, increasing expression from the cognate synthetic promoter by fivefold. Transgenic plants and non-transgenic plants were morphologically indistinguishable. The framework of this study can easily be adopted for other putative transcription factors to continue improvement of the plant synthetic biology toolbox.

Abstract B159: A first-in-human study of CDK9 inhibitor KB-0742 demonstrates evidence of tolerability and clinical activity

Abstract Background: Cyclin-dependent kinase 9 (CDK9) is a transcriptional regulator that mediates expression and downstream activity of oncogenic transcription factors (TFs) including MYC and chimeric TFs. Inhibiting CDK9 presents a promising approach to treat transcriptionally addicted cancers. KB-0742 is a potent, selective, orally bioavailable inhibitor of CDK9 that is currently being studied in a phase 1/2 dose escalation in solid tumors and NHL and cohort expansion for patients with transcription-dependent tumors (NCT04718675). Methods: KB-0742 is administered orally once daily for 3 consecutive days with 4 days off, weekly, in 28-day cycles until toxicity or disease progression. Eligibility criteria include age > 18 years, relapsed or refractory solid tumors or NHL, acceptable organ function and ECOG PS < 2. Study objectives include evaluation of safety, tolerability, PK, PD, and identification of KB-0742 MTD and RP2D using a modified Continuous Reassessment Method (mCRM). PK was measured from patient plasma and PD was assessed in peripheral blood mononuclear cells (PBMCs). PD measurements included analysis of phosphorylation of the CDK9 substrate serine 2 of the RNA Polymerase II C-terminal domain (pSER2), and changes in gene expression of prospectively defined CDK9 responsive genes. Results: As of 1 June 2023, 28 patients enrolled in dose escalation up to 60 mg. Patients had received a median of 4 (2-11) prior lines of therapy and remained on KB-0742 for a median of 86 days (10-311+). Twelve of 28 patients received at least 4 cycles of KB-0742. The most common tumor types enrolled were colorectal (5), chordoma (4), sarcoma (4), and breast (3). Treatment-emergent adverse events (TEAEs) occurring in >20% of patients included nausea, vomiting, anemia, fatigue, nervous system disorders, and peripheral edema. The most common reasons for treatment discontinuation were progressive disease, TEAEs, and withdrawal of consent. Across 4 dose levels, AUC and Cmax of KB-0742 increased linearly with a terminal half-life of 24 hours. At 60 mg, evidence of target engagement was observed by pSER2 reduction and proportional changes to CDK9 responsive genes. In the escalation phase 60 mg cohort, three patients, (2 colorectal and 1 PTCL) had MYC over-expressing tumors and achieved SD. Of the two patients with myxoid liposarcoma, both exhibited radiographic regression of their target lesions. One patient achieved 26% reduction in the sum of partial diameters lasting > 5 mos. The second patient achieved a RECIST 1.1 partial response and remains on treatment at > 12 mos. Consistent with the therapeutic hypothesis, both patients had TF fusions. Conclusions: KB-0742 treatment was well tolerated with manageable toxicity and no evidence of neutropenia. CDK9 inhibition was observed at the 60 mg dose level, and expansion cohorts in tumor types with a high prevalence of MYC amplification or overexpression and other transcriptionally addicted tumors are accruing. The MTD has not been reached and further dose escalation is continuing. Citation Format: Miguel Villalona-Calero, Monica Mita, Alain Mita, Noah Federman, Drew Rasco, David Spigel, Jia Luo, Glenn J Hanna, Gregory M Cote, Richard E Cutler, Pavan Kumar, Crystal MacKenzie, Charles Lin, Jorge F DiMartino, Elizabeth A Olek, Brian Van Tine. A first-in-human study of CDK9 inhibitor KB-0742 demonstrates evidence of tolerability and clinical activity [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B159.

Abstract 24 Development of an In Vitro B-Cell Differentiation Model for the Characterization of the Origin and Causation of ETV6::RUNX1+ Preleukemic Cells

Abstract Introduction Pregnant women are exposed to various carcinogenic substances that can subsequently affect their progeny. Recurrent preleukemic chromosomal translocations characteristic for childhood acute lymphoblastic leukemia (ALL) frequently emerge before birth in utero. The most common translocation t (12;21) occurs in 25% of B-cell precursor ALL and results in the formation of the chimeric oncogenic transcription factor ETV6::RUNX1. Objectives We aim to analyze factors promoting the generation of preleukemic ETV6::RUNX1 translocations, investigate the biological origin of these preleukemic cells, and develop an in vitro model of early B-cell differentiation to analyze the mechanisms underlying the persistence of ETV6::RUNX1+ preleukemic cells and leukemia evolution in children. Methods Genomic inverse PCR for exploration of ligated breakpoints (GIPFEL) (Schafer et. al., 2018) was applied to identify ETV6::RUNX1+ cord blood units (CBU). Screening results were validated by Sanger sequencing and quantified by real-time PCR (ipsogen ETV6-RUNX1 Kit, Qiagen). ETV6::RUNX1+ CBUs were thawed, sorted for CD34+ and CD19+ cells, and analyzed by qPCR. CD34+ were differentiated towards B-cell lineage on MSC feeder lines according to a modified protocol (Ichii et. al., 2008). Results We screened a cohort of 1405 cord bloods collected in 2004 and discovered that 103 (7.3%) carried the ETV6::RUNX1 fusion gene. The preleukemic translocation was detected in CD34+ stem and early B progenitor cells. For the establishment of an in vitro B-cell differentiation model, bone marrow (BM-MSCs) and CB (USSCs, CB-MSCs) stromal feeder cells were compared and the effect of different media (SCGM, DMEM/5 % FCS, MSCBM/10 % FCS) in the presence of stem cell factor (SCF), Flt-3, IL3, +/- IL7 was assessed. BM-MSCs demonstrated the highest ability to support B-lymphopoieses towards CD19+ cell differentiation in MSCBM media (~30%), primarily due to the release of IL-7 by the feeder layer. Currently, factors that may promote generation of preleukemic translocations in utero e.g. nitrosamines contained in food products, are being tested in this model. Discussion We identified the HSC/early B progenitor state as the origin of ETV6::RUNX1 translocations in CB. The establishment of an in vitro B-cell differentiation system will allow to test the impact of environmental carcinogens on the generation of ETV6::RUNX1+ pre-leukemic cells and their leukemic evolution.

A logical model of Ewing sarcoma cell epithelial-to-mesenchymal transition supports the existence of hybrid cellular phenotypes.

Ewing sarcoma (ES) is a highly aggressive pediatric tumor driven by the RNA-binding protein EWS (EWS)/friend leukemia integration 1 transcription factor (FLI1) chimeric transcription factor, which is involved in epithelial-mesenchymal transition (EMT). EMT stabilizes a hybrid cell state, boosting metastatic potential and drug resistance. Nevertheless, the mechanisms underlying the maintenance of this hybrid phenotype in ES remain elusive. Our study proposes a logical EMT model for ES, highlighting zinc finger E-box-binding homeobox 2 (ZEB2), miR-145, and miR-200 circuits that maintain hybrid states. The model aligns with experimental findings and reveals a previously unknown circuit supporting the mesenchymal phenotype. These insights emphasize the role of ZEB2 in the maintenance of the hybrid state in ES.

The N-Terminal Region of the BcWCL1 Photoreceptor Is Necessary for Self-Dimerization and Transcriptional Activation upon Light Stimulation in Yeast.

The BcWCL1 protein is a blue-light photoreceptor from the fungus Botrytis cinerea. This protein has a central role in B. cinerea circadian regulation and is an ortholog to WC-1 from Neurospora crassa. The BcWCL1 and WC-1 proteins have similar protein domains, including a LOV (Light Oxygen Voltage) domain for light sensing, two PAS (Per Arnt Sim) domains for protein-protein interaction, and a DNA binding domain from the GATA family. Recently, the blue-light response of BcWCL1 was demonstrated in a version without PAS domains (BcWCL1PAS∆). Here, we demonstrated that BcWCL1PAS∆ is capable of self-dimerization through its N-terminal region upon blue-light stimulation. Interestingly, we observed that BcWCL1PAS∆ enables transcriptional activation as a single component in yeast. By using chimeric transcription factors and the luciferase reporter gene, we assessed the transcriptional activity of different fragments of the N-terminal and C-terminal regions of BcWCL1PAS∆, identifying a functional transcriptional activation domain (AD) in the N-terminal region that belongs to the 9aaTAD family. Finally, we determined that the transcriptional activation levels of BcWCL1PAS∆ AD are comparable to those obtained with commonly used ADs in eukaryotic cells (Gal4 and p65). In conclusion, the BcWCL1PAS∆ protein self-dimerized and activated transcription in a blue-light-dependent fashion, opening future applications of this photoreceptor in yeast optogenetics.

Lentiviral vectors for inducible, transactivator-free advanced therapy medicinal products: Application to CAR-T cells

Controlling transgene expression through an externally administered inductor is envisioned as a potent strategy to improve safety and efficacy of gene therapy approaches. Generally, inducible ON systems require a chimeric transcription factor (transactivator) that becomes activated by an inductor, which is not optimal for clinical translation due to their toxicity. We generated previously the first all-in-one, transactivator-free, doxycycline (Dox)-responsive (Lent-On-Plus or LOP) lentiviral vectors (LVs) able to control transgene expression in human stem cells. Here, we have generated new versions of the LOP LVs and have analyzed their applicability for the generation of inducible advanced therapy medicinal products (ATMPs) with special focus on primary human Tcells. We have shown that, contrary to all other cell types analyzed, an Is2 insulator must be inserted into the 3' long terminal repeat of the LOP LVs in order to control transgene expression in human primary Tcells. Importantly, inducible primary Tcells generated by the LOPIs2 LVs are responsive to ultralow doses of Dox and have no changes in phenotype or function compared with untransduced Tcells. We validated the LOPIs2 system by generating inducible CAR-T cells that selectively kill CD19+ cells in the presence of Dox. In summary, we describe here the first transactivator-free, all-one-one system capable of generating Dox-inducible ATMPs.

Targeted long-read sequencing of the Ewing sarcoma 6p25.1 susceptibility locus identifies germline-somatic interactions with EWSR1-FLI1 binding

Ewing sarcoma (EwS) is a rare bone and soft tissue malignancy driven by chromosomal translocations encoding chimeric transcription factors, such as EWSR1-FLI1, that bind GGAA motifs forming novel enhancers that alter nearby expression. We propose that germline microsatellite variation at the 6p25.1 EwS susceptibility locus could impact downstream gene expression and EwS biology. We performed targeted long-read sequencing of EwS blood DNA to characterize variation and genomic features important for EWSR1-FLI1 binding. We identified 50 microsatellite alleles at 6p25.1 and observed that EwS-affected individuals had longer alleles (>135bp) with more GGAA repeats. The 6p25.1 GGAA microsatellite showed chromatin features of an EWSR1-FLI1 enhancer and regulated expression of RREB1, a transcription factor associated with RAS/MAPK signaling. RREB1 knockdown reduced proliferation and clonogenic potential and reduced expression of cell cycle and DNA replication genes. Our integrative analysis at 6p25.1 details increased binding of longer GGAA microsatellite alleles with acquired EWSR-FLI1 to promote Ewing sarcomagenesis by RREB1-mediated proliferation.

BET Bromodomain Degradation Disrupts Function but Not 3D Formation of RNA Pol2 Clusters.

Fusion-positive rhabdomyosarcoma (FP-RMS) is driven by a translocation that creates the chimeric transcription factor PAX3-FOXO1 (P3F), which assembles de novo super enhancers to drive high levels of transcription of other core regulatory transcription factors (CRTFs). P3F recruits co-regulatory factors to super enhancers such as BRD4, which recognizes acetylated lysines via BET bromodomains. In this study, we demonstrate that inhibition or degradation of BRD4 leads to global decreases in transcription, and selective downregulation of CRTFs. We also show that the BRD4 degrader ARV-771 halts transcription while preserving RNA Polymerase II (Pol2) loops between super enhancers and their target genes, and causes the removal of Pol2 only past the transcriptional end site of CRTF genes, suggesting a novel effect of BRD4 on Pol2 looping. We finally test the most potent molecule, inhibitor BMS-986158, in an orthotopic PDX mouse model of FP-RMS with additional high-risk mutations, and find that it is well tolerated in vivo and leads to an average decrease in tumor size. This effort represents a partnership with an FP-RMS patient and family advocates to make preclinical data rapidly accessible to the family, and to generate data to inform future patients who develop this disease.

Chromatin and Cancer: Implications of Disrupted Chromatin Organization in Tumorigenesis and Its Diversification.

A hallmark of cancers is uncontrolled cell proliferation, frequently associated with an underlying imbalance in gene expression. This transcriptional dysregulation observed in cancers is multifaceted and involves chromosomal rearrangements, chimeric transcription factors, or altered epigenetic marks. Traditionally, chromatin dysregulation in cancers has been considered a downstream effect of driver mutations. However, here we present a broader perspective on the alteration of chromatin organization in the establishment, diversification, and therapeutic resistance of cancers. We hypothesize that the chromatin organization controls the accessibility of the transcriptional machinery to regulate gene expression in cancerous cells and preserves the structural integrity of the nucleus by regulating nuclear volume. Disruption of this large-scale chromatin in proliferating cancerous cells in conventional chemotherapies induces DNA damage and provides a positive feedback loop for chromatin rearrangements and tumor diversification. Consequently, the surviving cells from these chemotherapies become tolerant to higher doses of the therapeutic reagents, which are significantly toxic to normal cells. Furthermore, the disorganization of chromatin induced by these therapies accentuates nuclear fragility, thereby increasing the invasive potential of these tumors. Therefore, we believe that understanding the changes in chromatin organization in cancerous cells is expected to deliver more effective pharmacological interventions with minimal effects on non-cancerous cells.

ESR1 fusions and therapeutic resistance in metastatic breast cancer.

Breast cancer is the most frequent female malignant tumor, and the leading cause of cancer death in women worldwide. The most common subtype of breast cancer is hormone receptor positive that expresses the estrogen receptor (ER). Targeting ER with endocrine therapy (ET) is the current standard of care for ER positive (ER+) breast cancer, reducing mortality by up to 40% in early- stage disease. However, resistance to ET represents a major clinical challenge for ER+ breast cancer patients leading to disease recurrence or progression of metastatic disease. Salient drivers of ET resistance are missense mutations in the ER gene (ESR1) leading to constitutive transcriptionalactivity and reduced ET sensitivity. These mutations are particularly prominent and deleterious in metastatic breast cancer (MBC). In addition to activating ESR1 point mutations, emerging evidence imposes that chromosomal translocation involving the ESR1 gene can also drive ET resistance through the formation of chimeric transcription factors with constitutive transcriptional activity. Although these ESR1 gene fusions are relatively rare, they are enriched in ET resistant metastatic disease. This review discusses the characteristics of ER fusion proteins and their association with clinical outcomes in more aggressive and metastatic breast cancer. The structure and classification of ER fusion proteins based on function and clinical significance are also addressed. Finally, this review summarizes the metastatic phenotypes exhibited by the ER fusion proteins and their role in intrinsic ET resistance.

IFT46 gene promoter-driven ciliopathy disease model in zebrafish.

Ciliopathies are human genetic disorders caused by abnormal formation and dysfunction of cellular cilia. Cilia are microtubule-based organelles that project into the extracellular space and transduce molecular and chemical signals from the extracellular environment or neighboring cells. Intraflagellar transport (IFT) proteins are required for the assembly and maintenance of cilia by transporting proteins along the axoneme which consists of complexes A and B. IFT46, a core IFT-B protein complex, is required for cilium formation and maintenance during vertebrate embryonic development. Here, we introduce transgenic zebrafish lines under the control of ciliated cell-specific IFT46 promoter to recapitulate human ciliopathy-like phenotypes. We generated a Tg(IFT46:GAL4-VP16) line to temporo-spatially control the expression of effectors including fluorescent reporters or nitroreductase based on the GAL4/UAS system, which expresses GAL4-VP16 chimeric transcription factors in most ciliated tissues during embryonic development. To analyze the function of IFT46-expressing ciliated cells during zebrafish development, we generated the Tg(IFT46:GAL4-VP16;UAS;nfsb-mCherry) line, a ciliated cell-specific injury model induced by nitroreductase (NTR)/metrodinazole (MTZ). Conditionally, controlled ablation of ciliated cells in transgenic animals exhibited ciliopathy-like phenotypes including cystic kidneys and pericardial and periorbital edema. Altogether, we established a zebrafish NTR/MTZ-mediated ciliated cell injury model that recapitulates ciliopathy-like phenotypes and may be a vertebrate animal model to further investigate the etiology and therapeutic approaches to human ciliopathies.

Engineering a two-gene system to operate as a highly sensitive biosensor or a sharp switch upon induction with β-estradiol

The human estrogen receptor has been used for about thirty years, in the yeast S. cerevisiae, as a component of chimeric transcription factors. Its ligand, β-estradiol, permits to control the protein translocation into the nucleus and, as a consequence, the expression of the gene(s) targeted by the synthetic transcription factor. Activators that are orthogonal to the yeast genome have been realized by fusing the human estrogen receptor to an activation and a DNA-binding domain from bacteria, viruses, or higher eukaryotes. In this work, we optimized the working of a β-estradiol-sensing device—in terms of detection range and maximal output signal—where the human estrogen receptor is flanked by the bacterial protein LexA and either the strong VP64 (from herpes simplex virus) or the weaker B42 (from E. coli) activation domain. We enhanced the biosensor performance by thoroughly engineering both the chimeric activator and the reporter protein expression cassette. In particular, we constructed a synthetic promoter—where transcription is induced by the chimeric activators—based on the core sequence of the yeast CYC1 promoter, by tuning parameters such as the length of the 5′ UTR, the distance between adjacent LexA binding sites (operators), and the spacing between the whole operator region and the main promoter TATA box. We found a configuration that works both as a highly sensitive biosensor and a sharp switch depending on the concentration of the chimeric activator and the strength of its activation domain.