Chromosomal Translocations Research Articles

Abstract 136: Determining the genomic basis of PAX3-FOXO1 mediated transformation in rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. Despite rigorous scientific advances and clinical trials, the survival rate for high-risk RMS has not increased above 20% in the last three decades. A deeper understanding of the basic biology driving RMS tumorigenesis is needed to discover novel therapeutic targets. RMS can be divided into two major histologic subtypes: embryonal RMS (ERMS) and alveolar RMS (ARMS). ARMS accounts for 20% of pediatric cases and has poorer survival. The majority of ARMS tumors harbor t(2;13)(q35;q14) or t(1;13)(p36;q14) chromosomal translocations resulting in the PAX3-FOXO1 (P3F) and PAX7-FOXO1 (P7F) fusion oncoproteins, respectively. ARMS tumors that lack oncofusions more closely resemble ERMS. Classifying on fusion status more accurately describes the disease and highlights the significance of P3/7F. ARMS tumor genomes only contain an average of 6.4 somatic mutations suggesting the oncofusion protein is the key oncogenic driver and a potential candidate for therapeutic intervention. RMS is commonly thought to arise from skeletal muscle progenitor cells that fail to terminally differentiate. However, RMS occurs throughout the body in sites devoid of skeletal muscle, underscoring the potential for alternative cells of origin. Our lab established that endothelial progenitors are indeed a cell of origin for ARMS by generating a genetically engineered mouse model with endothelial cell specific P3F expression coupled with Cdkn2a loss. We further generated a human model system derived from TP53-null human induced pluripotent stem cells (iPSCs), in which we direct differentiation to endothelial cell fate and then force P3F expression. P3F expression during endothelial cell directed differentiation blocked endothelial maturation and instead reprogrammed cells shifting cell fate to skeletal muscle-like cells that form ARMS tumors in mice. Generation of iPSC-derived ARMS (iARMS) occurs over 15 days and provides a unique model to determine the kinetics and specific genomic contributions facilitated by P3F during RMS transformation. To investigate the genomic changes occurring during iARMS transformation I utilized CUT&RUN for P3F and H3K27ac to assess P3F occupancy and enhancer landscape, ATAC-seq to assess chromatin accessibility, and RNA-seq to assess gene expression changes at timepoints throughout the transformation event. These results define the fundamental genomic changes needed for transformation into FP-RMS by P3F and uncovers the key determinants of ARMS cell identity. The iARMS system provides a unique platform to uncover how P3F establishes and maintains RMS cell state and to dissect the specific dependencies required to maintain the ARMS cell state. Understanding the fundamental mechanism of P3F-driven tumorigenesis could provide greater resolution into the key disease determinants to focus pre-clinical efforts on pivotal targets. Citation Format: Bradley T. Stevens, Yang Zhang, Randolph K. Larsen, Grace E. Adkins, Jack D. Hopkins, Darden W. Kimbrough, Matthew R. Garcia, Brian J. Abraham, Mark E. Hatley. Determining the genomic basis of PAX3-FOXO1 mediated transformation 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 136.

Abstract 4607: Targeted nanoparticle delivery of irinotecan and Ewing sarcoma-specific siRNA is an effective combination therapy for Ewing sarcoma

Abstract Background: Ewing sarcoma (ES) is an aggressive bone and soft tissue tumor affecting children and adolescents with an overall survival rate of 50%. Irinotecan (IR) is an effective chemotherapeutic agent but is associated with significant toxicity. Using an anti-CD99 targeted hybrid polymerized lipid nanoparticle containing IR, NV103, in pre-clinical mouse xenograft model of the disease, tumor ablation was achieved at 2mg/kg of IR versus 50mg/kg for free IR. All ES harbor a chromosomal translocation resulting in expression of a fusion EWS-FLI1 protein or equivalent. This chimeric gene is a transcriptional regulator that upregulates the FEZF1 and FEZF1-AS1 genes. Both EWS-FLI1 and FEZF1-AS1 genes are unique to ES and are not expressed in normal adult tissues. EWS-FLI1 promotes cell growth while the FEZF1-AS1 complex promotes tumor metastasis, a hallmark of ES. In vitro RNAi targeting of EF and FEZF1-AS1 results in tumor cell death. We hypothesized that combining EWS-FLI1 and/or FEZF1-AS1 targeted RNAi therapy with NV103 cytotoxic therapy would be more effective than any single modality therapy. Methods: NV103 was provided by NanoValent Pharmaceuticals. siRNA containing lipid nanoparticles (LNP-siRNA) were generated using PreciGenome NanoGenerator. siRNA was quantified using the ribogreen assay. Nanoparticles were analyzed for size and particle numbers by NanoSight. siRNA was used to target the following genes- EWS-FLI1, FEZF1, FEZF1-AS1, or GFP. A673 cells expressing GFP were used for this in vitro study. Various doses of IR and siRNA were used in combination and cell growth/death was imaged live using Incucyte. ATP assay was used to confirm live cells and to determine minimum effective dose (MED) over 72 hours of treatment. Results: LNP-siRNA, mean diameter 70nm and PDI<0.2, encapsulated 90% of the siRNA in solution. LNP are stable at 4°C for at least 4 weeks with minimal-to-no loss of activity. NV103 had a MED of 1µM. MED of various siRNA was 30 to 50nM. Combination of NV103 and LNP-siRNA reduced MED of IR to 250nM. One treatment dose of LNP-siRNA followed by NV103 treatment three days later maintained significant cell death over 10 days post treatment initiation. Control cells were 97% more confluent than NV103/EWS-FLI1 siRNA treated ones, 85% more than NV103/FEZF1 siRNA cells and 92% more than NV103/FEZF1-AS1 treated cells. siRNA combinations of EWS-FLI1 and FEZF1 or FEZF1-AS1 along with NV103 added no further benefit. Conclusion: Combination nanoparticle therapy of IR and ES-specific siRNA reduces IR MED by four-fold. LNP delivery of siRNA enables tumor-specific gene targeting. Most have no available small molecule therapy. Targeting ES cells with both a low dose (250nM) of IR and 30-50nM of siRNA against unique tumor biomarkers results in increased cell death with no IR toxicity. Further animal studies are required to determine the effectiveness of such therapy in vivo. Citation Format: Sheetal A. Mitra, Jean-Hugues Parmentier, Hyung-Gyoo Kang, Timothy J. Triche. Targeted nanoparticle delivery of irinotecan and Ewing sarcoma-specific siRNA is an effective combination therapy for 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 4607.

Abstract 1723: Targeting SMARCA1 in rhabdomyosarcoma: Potential therapeutic implications

Abstract Background: Rhabdomyosarcoma (RMS) is the most common soft tissue in pediatric sarcoma, and studies demonstrate that RMS arises from skeletal muscle precursor cells. RMS, genetically divided into two subtypes: PAX-FOXO1 fusion positive RMS (FP-RMS), which is driven by chromosomal translocation involving PAX3 or PAX7 genes with FOXO1 and PAX-FOXO1 fusion negative RMS (FN-RMS), which is marked by mutations in RAS isoforms and some other genes. Chromatin was one of the earliest identified targets for cancer therapy. Several chromatin remodeling proteins are associated with cancer progression processes such as proliferation, differentiation, apoptosis, and tumorigenesis. Components of chromatin remodeling complexes have been classified as both oncogenes and tumor suppressors. The ISWI family protein, SMARCA1 has been implicated in tumorigenesis for several cancer types. ISWI complexes regulate cell differentiation and proliferation in other cell systems, but their impact on myogenesis is not well understood. In this study, we will characterize the function of SMARCA1 in RMS cells and skeletal muscle. We hypothesize that SMARCA1 acts to modulate chromatin accessibility and drive RMS tumorigenic growth. Methods: We employed RNA-seq, ATAC-seq, and CUT&RUN to study the impact of SMARCA1 in RMS cells. Furthermore, phenotypic experiments were performed to determine the influence of SMARCA1 on differentiation and proliferation. Results: SMARCA1 is expressed at a high level in RMS tissues but not in muscle tissues. Our findings show that SMARCA1 is modestly upregulated during differentiation in normal myogenesis. Upon treatment with trametinib, FP-RMS cell lines with SMARCA1 knockout exhibited a dramatic decrease in cell confluency and viability. Intriguingly, the control FP-RMS cell lines demonstrated increased SMARCA1 protein levels in response to trametinib, which could suggest a resistance mechanism to the treatment. Trametinib also affected SMARCA1 mRNA expression in FN-RMS cells. Additionally, it was discovered that the elimination of SMARCA1 not only resulted in a reduction in cell migration but also led to a failure of these cells to develop spheres, unlike the control cells, where the migration gap healed in the usual manner and the spheres were observed. These findings may align with our hypothesis that SMARCA1 regulates chromatin accessibility, potentially impacting adhesion properties and signaling pathways. Analysis of gene clusters in RMS transcriptomic data indicated that SMARCA1 co-expresses with a RAS effector, RALA. SMARCA1 is also clustered with HDAC2. In addition, SMARCA1 interacted with HDAC2 in RMS cells, as determined by co-immunoprecipitation experiments. Conclusions: Our findings suggest that SMARCA1 may influence RMS tumor growth. This study might contribute to a deeper understanding of SMARCA1's role in RMS and support its credentialing as a novel therapeutic avenue. Citation Format: Ashwaq Aljabri, Katie Hebron, Yuliya Kriga, Juan Manuel Caravaca, Aiysha Althobaiti, Stacey Stauffer, Angela Kim, Lauren Stoak, Morgan Porter, Jyoti Shetty, Bao Tran, Matthew Geisler, Judith Davie, Marielle Yohe. Targeting SMARCA1 in rhabdomyosarcoma: Potential therapeutic implications [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 1723.

Abstract 6062: Potent and orally bioavailable BCL6 protein degraders demonstrate efficacy in pre-clinical models of diffuse large B-cell lymphoma (DLBCL)

Abstract The B-cell lymphoma 6 (BCL6) transcription repressor protein is a protooncogene especially in diffuse large B-cell lymphoma (DLBCL), conferring survival, protection, and maintenance of lymphoma cells. BCL6 expression in normal B cells contributes to initiation and maintenance of the germinal centers (GC). Somatic mutations of the BCL6 gene including chromosome translocations have been shown to result in the deregulation of BCL6 expression and contributes to a subgroup of poor prognostic double- and triple-hit lymphomas. To solve this unmet clinical need, we have developed highly specific, potent, and orally bioavailable BCL6 protein degrader HSK43608 that demonstrates potent in vitro and in vivo efficacy in multiple pre-clinical DLBCL models. HSK43608 achieves >90% BCL6 degradation at 10 nM and DC50 of <1 nM in the OCI-Ly1 model following 24 hr treatment. Time-course studies showed >90% BCL6 degradation within 1 hr which could be maintained at 24 hrs. Importantly, medicinal chemistry efforts have resulted in the successful development of new-generation IMIDs to improve the oral bioavailability of BCL6 degrader for in vivo treatment. As expected, concentration of HSK43608 in tumor tissue was much higher than that in plasma in the DLBCL xenograft models. This tumor tissue enrichment of HSK43608 achieved >95% BCL6 degradation 48 hr after last dosing and convincing tumor growth inhibition. It is worth noting that BCL6 knockout mice suffer from myocarditis with eosinophils infiltration in heart muscle. The tissue distribution study demonstrated less HSK43608 in heart than in plasma, partially explained why there was no sign of myocarditis with eosinophils infiltration in the heart of the HSK43608 treated mice. Taking advantage of the new IMIDs structure, HSK43608 barely induce the degradation of the first generation IMIDs targets including GSPT1, SALL4, CK1α, Aiolos and Ikaros, indicating its safety. In summary, these results indicate HSK43608 as a potent and orally bioavailable BCL6 degrader and support further development. Citation Format: Jun Xu, Yangguang Li, Ju Wang, Pingming Tang, Yuting Liao, Chen Zhang, Pangke Yan. Potent and orally bioavailable BCL6 protein degraders demonstrate efficacy in pre-clinical models of diffuse large B-cell lymphoma (DLBCL) [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 6062.

Abstract 3306: RGT-M001, a first-in-class small molecule mRNA degrader of the oncogenic transcription factor c-Myb, demonstrated remarkable single agent anti-tumor efficacy in cancer patient-derived xenograft model

Abstract Dysregulated transcription factors (TFs) represent a unique class of drug targets that drive aberrant gene expression programs involved in the major hallmarks of cancers. However, developing drug-like small molecules targeting TF proteins for clinical purposes has proven to be extremely challenging. Here, we present a novel and innovative approach to degrade the RNA transcript of the master oncogenic transcription factor C-MYB, thereby preventing its undesirable expression, using potent and orally available small molecules. C-MYB is a transcription factor that regulates differentiation and proliferation programs in normal cells, including hematopoietic, stem and epithelial cells. In numerous cancer types, such as adenoid cystic carcinoma (ACC), leukemia, colorectal cancer, and breast cancer, C-MYB is a well-established oncogenic transcription factor, overactivated via different mechanisms, including chromosomal translocations and gene amplification. Here, we leveraged our integrative RNA-targeting platform to identify and validate actionable cryptic exon target sites in the human C-MYB gene. We identified RGT-M001, a potent small molecule that can selectively induce the inclusion of the cryptic exon into the final C-MYB transcripts, resulting in a robust decrease of C-MYB canonical transcripts and C-MYB protein in cells. In a functional assay, we demonstrated that RGT-M001 has potent cell-killing activity against a large panel of cancer cell lines overexpressing C-MYB (EC50 ~ 20 - 300nM), while sparing normal cells. To confirm RGT-M001's on-target effect, we demonstrated a robust correlation between RGT-M001 cell-killing activity and the knockdown of C-MYB RNA and protein. We further investigated the anti-tumor activity of RGT-M001 in an ACC patient-derived xenograft (PDx) mouse model harboring C-MYB-NFBI fusion. Recurrent or metastatic ACC is a malignant neoplasm of predominantly salivary gland origin for which effective approved therapies are lacking; the best reported ORR being 15.6% for lenvatinib. As a single agent, RGT-M001 reduced in vivo C-MYB transcript levels by >80% at peak drug exposure and induced a remarkable tumor growth inhibition response (~70% TGI) in the ACCX11 PDx mouse model, surpassing the therapeutic benchmark Lenvatinib (40% TGI). Importantly, the RGT-M001 regimen was well tolerated. Finally, we showed that the combination of RGT-M001 with the Notch Inhibitor AL-101 resulted in complete inhibition of tumor growth. In conclusion, these data demonstrate that small molecules targeting RNA are a safe and effective approach to address previously undruggable protein targets. Down-regulation of C-MYB by our RNA-targeting small molecules is an attractive therapeutic strategy to treat ACC and other cancers driven by C-MYB dysregulations. Citation Format: Norman Lu, Patricia Soulard, Kay Li, Heather Sadlish, Chris Yates, Xiubin Gu, Ibrahim Kays, Jae Lee, Sam Hasson, Zhiping Weng, Simon Xi, Travis Wager. RGT-M001, a first-in-class small molecule mRNA degrader of the oncogenic transcription factor c-Myb, demonstrated remarkable single agent anti-tumor efficacy in cancer patient-derived xenograft model [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 3306.

Abstract 3165: Targeting angiogenesis for improved survival in mantle cell lymphoma

Abstract Mantle cell lymphoma (MCL) is a subtype comprising 6% of all NHL cases and is characterized by overexpression of cyclin D1 due to chromosomal translocation t(11;14) in the nodal MCL subtype. One of the best discriminatory genes between these conventional and indolent MCL tumors is SOX11; in addition, SOX11 has been shown to promote angiogenesis in MCL. Further SOX11 protein expression in MCL patients correlated with increased microvascular density and poor outcomes. Bulk transcriptomics analysis was performed on MCL patients (n=24) and CD19-purified B cells from healthy donors (HD,n=3). To understand the role of the angiogenic process in MCL, we queried 141 genes related to angiogenesis (GO:0001525). Our analysis showed that many genes involved in angiogenesis were predominantly overexpressed in MCL compared to HD, including FLT1 and KDR (p-value;0.05; FC>2). SOX11 has been shown to regulate PDGFA in MCL cell lines. Consistent with this observation, we also see upregulated PDGFA levels in MCL patients. However, PDGFA upregulation was not statistically significant in our analysis. Further expression of VEGFR2 is overexpressed in MCL Patients (n=122) compared to naïve or activated-B cells from HD. We validated that VEGFR2 protein levels were higher in MCL primary patients than in healthy donor PBMC. Further, in two independent cohorts, high VEGFR2 expression shows significant adverse effects on 5-year overall survival (OS). To explore the potential of targeting VEGFR, we utilized lucitanib, which potently and selectively inhibits VEGFRs. Treatment with lucitanib resulted in a dose-dependent decrease in cell viability and cellular death as determined by Annexin PI staining; further, MCL cells treated with lucitanib showed inhibition of autophosphorylation of VEGFR2 and demonstrated PARP cleavage associated with cytotoxicity in-vitro. To assess the role of VEGFR2 inhibitor lucitanib as a single agent in MCL growth in-vivo, we utilized two different murine models of MCLs. First, we used cell-line-derived xenograft (CDX) models of MCL where Jeko-1 and HS5 cells were injected together, followed by treatment (lucitanib;15mg/kg QOD; IP) and measured tumor volume until the ERC of any experimental mice was reached. Kaplan-Meier survival graphs were plotted, demonstrating that lucitanib treatment prolongs survival in-vivo. Further, to confirm if the lucitanib can provide an overall survival benefit in the systemic PDX MCL model, we injected the PDX cells via the tail vein in NSG mice and followed human-B2M levels for disease progression, and overall survival was the endpoint. Lucitanib treatment significantly prolonged median overall survival significantly in the systemic MCL model. Our data demonstrates that targeting angiogenesis in MCL can provide therapeutic benefits, as demonstrated in preclinical MCL models. Citation Format: Satishkumar Singh, Anuvrat sircar, Audrey Odier, Narendranath Epperla, Natarajan Muthusamy, Samir Parekh, Lalit Sehgal. Targeting angiogenesis for improved survival in mantle cell lymphoma [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 3165.

Abstract 987: Novel ALK mutations in EML4::ALK+ NSCLC resistant to TKIs identified by liquid biopsy

Abstract The identification of Anaplastic Lymphoma Kinase (ALK) chromosomal translocations led to the development of effective targeted therapies in a subset of Non-Small Cell Lung Cancer (NSCLC) patients. Several drugs are currently in clinical practice for advanced ALK+ NSCLC. However, resistance remains a challenge in these patients. The possibility to analyze circulating tumor DNA (ctDNA) with non-invasive methods can greatly improve prognostic capabilities, by allowing early detection of relapse and of drug-resistant mutations from peripheral blood. We report a series of 15 consecutive relapsed NSCLC patients (12 ALK+, 3 ROS1+) treated at the Fondazione IRCCS-San Gerardo dei Tintori (Monza, Italy) that were investigated by liquid biopsy at tyrosine kinase inhibitor (TKI) failure, through amplicon deep sequencing of the ALK/ROS1 kinase domains. Median DNA yield was 4.8 ng per ml of plasma and mean fragment size was 176 bp. Mutational hotspot coding regions of ALK and ROS1 genes were amplified by high-fidelity PCR and sequenced. Mean coverage of called variants was 24,687x. The threshold for mutation calling was set at 0.6%, after correcting for mapping quality, minimum coverage, and strand bias. Analysis of background signal on healthy control DNA identified 0.0024 mismatches per mapped base. Overall, plasma ctDNA analysis detected an ALK mutation in 7/18 relapse samples from 7/15 patients. No ROS1 mutations were found in ROS1 fusion-positive patients. Among 12 patients carrying an EML4::ALK fusion, 10 variants potentially driving resistance were found. Along with mutations that were previously identified (L1196M/G1202R, E1154K, F1174L), novel ALK variants were detected such as L1198R, T1211I, C1235R, C1237Y, L1196P. Mutations were characterized for sensitivity to ALK inhibitors in a BaF3-EML4::ALK cell model, using cell proliferation assays and western blotting to evaluate the effects on ALK phosphorylation. C1237Y and L1196P mutants appeared to resist all TKIs; hence, we further tested their sensitivity to novel investigational drugs, zotizalkib and repotrectinib. Both mutants demonstrated resistance, suggesting that these two mutations confer broad resistance to TKIs. Molecular modelling of C1237Y and L1196P mutants was run to elucidate the mechanisms of resistance. The ADP-C1237Y complex showed a much smaller root-mean-square deviation than ADP-WT, indicating a higher affinity/activity of the mutant. L1196 interacts hydrophobically with a methyl group of lorlatinib, P1196 is predicted to lose such interaction, while ATP binding is not affected. The presence of C1237Y and L1196P variants, is a relevant finding due to their resistance, even after the exposure to the fourth-generation inhibitor zotizalkib. The clonal selection of these mutations, made by second/third generation ALK TKIs may lead to a complete refractory status, which would then render TKIs useless in a sequential therapeutic strategy. Citation Format: Federica Malighetti, Matteo Villa, Elisa Sala, Geeta Sharma, Giulia Arosio, Maria Gemelli, Chiara Manfroni, Diletta Fontana, Nicoletta Cordani, Raffaella Meneveri, Alfonso Zambon, Rocco Piazza, Fabio Pagni, Diego Cortinovis, Luca Mologni. Novel ALK mutations in EML4::ALK+ NSCLC resistant to TKIs identified by liquid biopsy [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 987.

Abstract 6234: Single-cell analysis of state-transition from health to leukemia and the leukemic cell heterogeneities in inv(16) acute myeloid leukemia (AML)

Abstract AML is an aggressive hematological malignancy with heterogeneous genetic abnormalities. Using a conditional knock-in mouse model (Cbfb56M/+/Mx1-Cre) to mimic the somatic acquisition of fusion gene CBFB-MYH11 (CM), created in the recurrent chromosome translocation inv(16)(p13q22) of AML, we previously reported that the blood transcriptome undergoes state-transition from health to leukemia. In this study, we analyze the transcriptomic alterations and leukemic cell heterogeneities in a leukemia state-space using single-cell RNA sequencing (scRNA-seq). CM expression was induced in Cbfb56M/+/Mx1-Cre mice by poly (I:C) injection, and scRNA-seq analysis was performed on peripheral blood mononuclear cells (PBMC) and bone marrow (BM) cells from leukemic mice and age-matched wild-type (WT) controls. More than 105,000 cells were subjected to the following dimensionality reduction and cell type annotation. By performing principal component analysis (PCA), we observed a Y-shape cell distribution with emergence of a divergent branch occupied by leukemic cells which can be mapped to a leukemic state. The principal components 1 and 2 (PC1 and PC2) clearly distinguish the leukemic cells from normal cell types such as myeloid cells and lymphocytes. By accessing the eigenvalue of genes for PC1 and PC2, we quantified the crucial genes and pathways related to leukemia progression at a single-cell level. We characterized cKit+ leukemic cell clusters by transcriptome-based correlation and gene expression pattern and found that they resembled stem cells and megakaryocytic-erythroid progenitors (MEP) with dysregulated pathways, including upregulated "inflammatory response" and downregulated pathways related to Myc targets, oxidative phosphorylation, mitochondrial protein translation, and ribosomal biogenesis/assembly. Examining the 31 clusters (C0-C30) seen in cKit+ BM cells, we identified 9 leukemic clusters exhibiting distinct features, including high heme-metabolism (C0, C1, and C2), high proliferation (C1, C8), S phase (C4, C10), low oxidative phosphorylation (C2, C5, C17), high Cd9 (C10), high Egfl7 (C7, C8). In conclusion, our study unveils state-transition from health to leukemia at single-cell resolution, and uncovers diverse biological heterogeneities associated with the inv(16) AML state. Further refinement of transcriptome state-transition and single-cell analysis will provide an analytical framework for modeling the dynamic changes during disease evolution at the single-cell level. Citation Format: Yu-Hsuan Fu, Lianjun Zhang, Ying-Chieh Chen, David E. Frankhouser, Lisa Uechi, Denis O'Meally, Sergio Branciamore, Guido Marcucci, Russell Rockne, Ya-Huei Kuo. Single-cell analysis of state-transition from health to leukemia and the leukemic cell heterogeneities in inv(16) acute myeloid leukemia (AML) [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 6234.

Abstract 405: Translocation detection in cancer using low-pass pore-c sequencing

Abstract Complex chromosomal rearrangements, including translocations, play a critical role in oncogenesis and are often identified as recurrent genetic aberrations in hematologic malignancies and solid tumors[1]. Translocation detection by karyotyping of an individual’s metaphase chromosomes remains challenging for detection of balanced rearrangements, which have no gain or loss of genetic material. Rearrangement detection using whole-genome sequencing is a viable alternative, but it relies on breakpoint-spanning reads and requires high sequencing depth (30-60x) with long reads to achieve high sensitivity, especially in repetitive regions of the genome[2]. Here, we describe a workflow for translocation detection at low sequencing depth with Oxford Nanopore’s long read chromatin conformation capture technique, Pore-C[3]. Importantly, for translocation calling, Pore-C does not rely on breakpoint-spanning reads but rather the high intrachromosomal interaction frequency of genomic regions around the breakpoint, thus largely lowering sequencing depth requirements and reducing mapping issues in low complexity regions. We prepared, barcoded, and sequenced Pore-C libraries of 3 cancer cell lines including lung cancer (A549), Acute Monocytic Leukemia (THP-1), melanoma (COLO 829) and its matching normal (COLO 829 BL) on a single Q20+ MinION flow cell (FLO-MIN114). Genome-wide contact maps of low-pass data (<1.5 Gbps per sample) were compared against higher depth (>30 Gbps per sample) Hi-C maps [4], revealing that low-pass Pore-C successfully captured large-scale genomic rearrangement. For example, chromosomal pairs chr8-chr11 and chr15-chr19 in A549, chr9-chr11 and chr1-chr20 in THP1, and chr7-chr15 in Colo829 were detected at <0.5X depth of sequencing coverage. These translocation events were further validated by breakpoint analysis using adaptive sampling, where targeted regions were previously confirmed by independent studies[4],[5]. Low-pass Pore-C detects translocations in an unbiased manner and does not require prior knowledge of the translocation structure. Combined with its simple sample-preparation workflow and the capability to provide genome-wide copy-number information in a single experiment, it can serve as a cost-effective and comprehensive tool for cancer genomic studies. [1] Chromosomal translocations in human cancer, Nature, 372, 143 (1994) [2] Hi-C as a tool for precise detection and characterisation of chromosomal rearrangements and copy number variation in human tumors, Genome Biology, 18, 125 (2017) [3] Identifying synergistic high-order 3D chromatin conformations from genome-scale nanopore concatemer sequencing, Nature Biotechnology, 40, 1488(2022) [4] Chromosomal translocations detection in cancer cells using chromosomal conformation capture data, Genes (Basel), 13, 7 (2022) [5] A multi-platform reference for somatic structural variation detection, Cell Genomics, 2, 6 (2022) Citation Format: Scott Hickey, Xiaoguang Dai, Sergey Aganezov, John Beaulaurier, Eoghan Harrington, Sissel Juul. Translocation detection in cancer using low-pass pore-c sequencing [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 405.

Abstract 266: The effects of retinoid agents and retinoic acid receptor genotype on proliferation, differentiation, and stem cells in colorectal cancer

Abstract Understanding how to therapeutically target colonic stem cells (SCs) is important because SC overpopulation drives CRC growth and development. Our goal is to therapeutically sensitize cancer SCs (CSCs) to the differentiation-inducing effects of retinoid agents. We previously found that ALDH specifically marks normal and malignant colonic SCs. We also found that retinoic acid (RA) receptors and other RA signaling components are selectively expressed in ALDH+ SCs, indicating that RA signaling occurs through ALDH+ SCs. Because all-trans retinoic acid (ATRA) is a highly effective treatment for acute promyelocytic leukemia, which is caused by a genetic translocation involving retinoic acid receptor alpha (RARA), we conjecture that the response of RA agents may be effective against solid tumors depending on their pathway genotype. Our bioinformatics analysis on mutations and overexpression of RA signaling component genes in CRC showed that most of the RA pathway genes are altered in human CRCs. Therefore, we hypothesize that the ability of retinoid agents and retinoid metabolism blocking agents to induce differentiation of CSCs depends on the RA pathway genotype of CRC cells.Testing the effect of various retinoid drugs on cell proliferation shows that RA pathway genotype affects the response of RA agents in CRC cell lines. HCT116 and SW480 CRC cell lines, which have RA receptor mutations, displayed resistance to ATRA. In contrast, HT29 CRC cells that have wild-type RA receptors are sensitive to ATRA. All three cell lines however showed similar dose responses to the ATRA metabolizing enzyme, CYP26A1 inhibitor Liarozole. This finding indicates that regardless of RA receptor mutations, inhibition of ATRA metabolism can increase intracellular RA levels in a similar manner on all three cell lines to induce anti-proliferative effects. We used CRISPR-Cas9 gene editing to knock out the RARA gene in HT29 cells and evaluated its response to ATRA compared to HT29 cells. The RARA knockout derived lines showed reduced sensitivity to ATRA treatment and a slower rate of cell division compared to HT29 control cells. Additionally, Nanostring mRNA profiling analysis shows that ATRA treatment of HT29 cells increases expression of RARA, CYP26A1, and KRT20 (differentiation marker); and decreased decreases expression of CSC markers LGR5 and ALDH1A1. Altogether, these findings indicate that upregulated RA signaling can reduce cell proliferation, increase differentiation, and decrease CSC numbers in CRCs. Overall, our findings indicate that colon SCs are regulated by RA signaling mechanisms and dysregulation of RA signaling contributes to the SC overpopulation that drives CRC growth. Thus, therapeutically sensitizing CSCs to differentiation-inducing effects of retinoid agents should provide insight into designing new SC-targeted therapies for CRC. Citation Format: Victoria O. Hunsu, Caroline O. Facey, Bruce M. Boman. The effects of retinoid agents and retinoic acid receptor genotype on proliferation, differentiation, and stem cells in colorectal cancer [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 266.

The histone acetyltransferase KAT6B is required for hematopoietic stem cell development and function

The histone lysine acetyltransferase KAT6B (MYST4, MORF, QKF) is the target of recurrent chromosomal translocations causing hematological malignancies with poor prognosis. Using Kat6b germline deletion and overexpression in mice, we determined the role of KAT6B in the hematopoietic system. We found that KAT6B sustained the fetal hematopoietic stem cell pool but did not affect viability or differentiation. KAT6B was essential for normal levels of histone H3 lysine 9 (H3K9) acetylation but not for a previously proposed target, H3K23. Compound heterozygosity of Kat6b and the closely related gene, Kat6a, abolished hematopoietic reconstitution after transplantation. KAT6B and KAT6A cooperatively promoted transcription of genes regulating hematopoiesis, including the Hoxa cluster, Pbx1, Meis1, Gata family, Erg, and Flt3. In conclusion, we identified the hematopoietic processes requiring Kat6b and showed that KAT6B and KAT6A synergistically promoted HSC development, function, and transcription. Our findings are pertinent to current clinical trials testing KAT6A/B inhibitors as cancer therapeutics.

Current Progress on the Influence Human Genetics Has on the Efficacy of Tyrosine Kinase Inhibitors Used to Treat Chronic Myeloid Leukemia.

The use of tyrosine kinase inhibitors (TKIs) has become the mainstay of treatment in patients suffering from chronic myeloid leukemia (CML), an adult leukemia caused by a reciprocal translocation between chromosomes 9 and 22, which createsan oncogene resulting in a myeloproliferative neoplasm. These drugs function by inhibiting the ATP-binding site on the fusion oncoprotein andsubsequently halting proliferative activity. The goal of this work is to investigate the current state of research into genetic factors that influence the efficacy of four FDA-approved TKIs used to treat CML. This overview attempts to identify genetic criteria that could be considered when choosing one drug over the others and to identify where more research is needed. Our results suggest thatthe usual liver enzymes impacting patient response may not be a major factor affecting the efficacy of imatinib, nilotinib, and bosutinib, and yet, that is where most of the past research has focused. More research is warranted on the impact that human polymorphisms of the CYP enzymes have on dasatinib. The impact of polymorphisms in UGT1A1 should be investigated thoroughly in other TKIs, not only nilotinib.The role of influx and efflux transporters has been inconsistent thus far, possibly due to failures to account for the multiple proteins that can transport TKIs and the impact that tumors have on transporter expression. Because physicians cannot currently use a patient's genetic profile to better target their treatment withTKIs, it is critical that more research be conducted on auxiliary pathways or off-target binding effects to generate new leads for further study. Hopefully, new avenues of research will help explain treatment failures and improve patient outcomes.

The molecular signature of BCR::ABLP210 and BCR::ABLT315I in a Drosophila melanogaster chronic myeloid leukemia model

Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disorder resulting from a balanced translocation leading to BCR::ABL1 oncogene with increased tyrosine kinase activity. Despite the advancements in the development of tyrosine kinase inhibitors (TKIs), the T315I gatekeeper point mutation in the BCR::ABL1 gene remains a challenge. We have previously reported in a Drosophila CML model an increased hemocyte count and disruption in sessile hemocyte patterns upon expression of BCR::ABL1p210 and BCR::ABL1T315I in the hemolymph. In this study, we performed RNA sequencing to determine if there is a distinct gene expression that distinguishes BCR::ABL1p210 and BCR::ABL1T315I. We identified six genes that were consistently upregulated in the fly CML model and validated in adult and pediatric CML patients and in a mouse cell line expressing BCR::ABL1T315I. This study provides a comprehensive analysis of gene signatures in BCR::ABL1p210 and BCR::ABL1T315I, laying the groundwork for targeted investigations into the role of these genes in CML pathogenesis.

The Screening of microRNAs in Chronic Myeloid Leukemia: A Clinical Evaluation.

Chronic myeloid leukemia (CML) is a type of leukemia whose main genetic marker is the reciprocal translocation that leads to the production of the BCR::ABL1 oncoprotein. The expression of some genes may interfere with the progression and development of leukemias. MicroRNAs are small non-coding RNAs that have the potential to alter the expression of some genes and may be correlated with some types of leukemia and could be used as biomarkers in the diagnosis and prognosis of patients. Therefore, this project carried out an analysis of microRNA-type plasma biomarkers in patients with chronic myeloid leukemia at unique points, including follow-up analysis of patients from the Erasto Gaertner Hospital. 35 microRNAs were analyzed in different cohorts. Inside those groups, 70 samples were analyzed at unique points and 11 patients in a follow-up analysis. Statistically different results were found for microRNA-7-5p, which was found to be upregulated in patients with high expression of the BCR::ABL1 transcript when compared to healthy controls. This microRNA also had evidence of behavior related to BCR::ABL1 when analyzed in follow-up, but strong evidence was not found. In this way, this work obtained results that may lead to manifestations of a relationship between miR-7-5p and chronic myeloid leukemia, and evaluations of possible microRNAs that are not related to this pathology.

Identification of a rare copy number polymorphic gain at 3q12.2 with candidate genes for familial endometriosis.

Endometriosis is a complex disease that affects 10-15% of women of reproductive age. Familial studies show that relatives of affected patients have a higher risk of developing the disease, implicating a genetic role for this disorder. Little is known about the impact of germline genomic copy number variant (CNV) polymorphisms on the heredity of the disease. In this study, we describe a rare CNV identified in two sisters with familial endometriosis, which contain genes that may increase the susceptibility and progression of this disease. We investigated the presence of CNVs from the endometrium and blood of the sisters with endometriosis and normal endometrium of five women as controls without the disease using array-CGH through the Agilent 2x400K platform. We excluded common CNVs that were present in the database of genomic variation. We identified, in both sisters, a rare CNV gain affecting 113kb at band 3q12.2 involving two candidate genes: ADGRG7 and TFG. The CNV gain was validated by qPCR. ADGRG7 is located at 3q12.2 and encodes a G protein-coupled receptor influencing the NF-kappaβ pathway. TFG participates in chromosomal translocations associated with hematologic tumor and soft tissue sarcomas, and is also involved in the NF-kappa B pathway. The CNV gain in this family provides a new candidate genetic marker for future familial endometriosis studies. Additional longitudinal studies of affected families must confirm any associations between this rare CNV gain and genes involved in the NF-kappaβ pathway in predisposition to endometriosis.

Impact of NOTCH1 expression in primary breast adenoid cystic carcinoma

AimsAdenoid cystic carcinoma (AdCC) originates from salivary-type like glands in the head and neck, lung, and breast. AdCC shows chromosomal translocation, resulting in MYB::NFIB fusion and overexpression of MYB. Recently,...

Application of optical genome mapping technology for the detection of chromosomal structural variations

To assess the value of optical genome mapping (OGM) for the detection of chromosomal structural abnormalities including ring chromosomes, balanced translocations, and insertional translocations. Clinical data of four patients who underwent pre-implantation genetic testing concurrently with OGM and chromosomal microarray analysis at the Center of Reproductive Medicine of the Sixth Affiliated Hospital of Sun Yat-sen University from January to October 2022 due to chromosomal structural abnormalities were selected as the study subjects. Some of the results were verified by multi-color fluorescence in situ hybridization. The OGM has successfully detected a balanced translocation and fine mapped the breakpoints in a patient. Among two patients with insertional translocations, OGM has provided more refined breakpoint locations than karyotyping analysis in a patient who had chromosome 3 inserted into chromosome 6 and determined the direction of the inserted fragment. However, OGM has failed to detect the chromosomal abnormality in a patient with chromosome 8 inserted into the Y chromosome. It has also failed to detect circular signals in a patient with ring chromosome mosaicism. OGM has successfully detected chromosomal structural variations in the four patients and provided assistance for their diagnosis.

On- and off-target effects of paired CRISPR-Cas nickase in primary human cells

Undesired on- and off-target effects of CRISPR-Cas nucleases remain a challenge in genome editing. While the use of Cas9 nickases has been shown to minimize off-target mutagenesis, their use in therapeutic genome editing has been hampered by a lack of efficacy. To overcome this limitation, we and others have developed double-nickase-based strategies to generate staggered DNA double-strand breaks to mediate gene disruption or gene correction with high efficiency. However, the impact of paired single-strand nicks on genome integrity has remained largely unexplored. Here, we developed a novel CAST-seq pipeline, dual CAST, to characterize chromosomal aberrations induced by paired CRISPR-Cas9 nickases at three different loci in primary keratinocytes derived from patients with epidermolysis bullosa. While targeting COL7A1, COL17A1, or LAMA3 with Cas9 nucleases caused previously undescribed chromosomal rearrangements, no chromosomal translocations were detected following paired-nickase editing. While the double-nicking strategy induced large deletions/inversions within a 10 kb region surrounding the target sites at all three loci, similar to the nucleases, the chromosomal on-target aberrations were qualitatively different and included a high proportion of insertions. Taken together, our data indicate that double-nickase approaches combine efficient editing with greatly reduced off-target effects but still leave substantial chromosomal aberrations at on-target sites.

Optical Genome Mapping as a Potential Routine Clinical Diagnostic Method.

Chromosome analysis (CA) and chromosomal microarray analysis (CMA) have been successfully used to diagnose genetic disorders. However, many conditions remain undiagnosed due to limitations in resolution (CA) and detection of only unbalanced events (CMA). Optical genome mapping (OGM) has the potential to address these limitations by capturing both structural variants (SVs) resulting in copy number changes and balanced rearrangements with high resolution. In this study, we investigated OGM's concordance using 87 SVs previously identified by CA, CMA, or Southern blot. Overall, OGM was 98% concordant with only three discordant cases: (1) uncalled translocation with one breakpoint in a centromere; (2) uncalled duplication with breakpoints in the pseudoautosomal region 1; and (3) uncalled mosaic triplication originating from a marker chromosome. OGM provided diagnosis for three previously unsolved cases: (1) disruption of the SON gene due to a balanced reciprocal translocation; (2) disruption of the NBEA gene due to an inverted insertion; (3) disruption of the TSC2 gene due to a mosaic deletion. We show that OGM is a valid method for the detection of many types of SVs in a single assay and is highly concordant with legacy cytogenomic methods; however, it has limited SV detection capabilities in centromeric and pseudoautosomal regions.

Rapid and cost-effective molecular karyotyping in wheat, barley, and their cross-progeny by chromosome-specific multiplex PCR

BackgroundInterspecific hybridisation is a powerful tool for increasing genetic diversity in plant breeding programmes. Hexaploid wheat (Triticum aestivum, 2n = 42) × barley (Hordeum vulgare, 2n = 14) intergeneric hybrids can contribute to the transfer of agronomically useful traits by creating chromosome addition or translocation lines as well as full hybrids. Information on the karyotype of hybrid progenies possessing various combinations of wheat and barley chromosomes is thus essential for the subsequent breeding steps. Since the standard technique of chromosome in situ hybridisation is labour-intensive and requires specific skills. a routine, cost-efficient, and technically less demanding approach is beneficial both for research and breeding.ResultsWe developed a Multiplex Polymerase Chain Reaction (MPCR) method to identify individual wheat and barley chromosomes. Chromosome-specific primer pairs were designed based on the whole genome sequences of ‘Chinese Spring’ wheat and ‘Golden Promise’ barley as reference cultivars. A pool of potential primers was generated by applying a 20-nucleotide sliding window with consecutive one-nucleotide shifts on the reference genomes. After filtering for optimal primer properties and defined amplicon sizes to produce an ordered ladder-like pattern, the primer pool was manually curated and sorted into four MPCR primer sets for the wheat A, B, and D sub-genomes, and for the barley genome. The designed MPCR primer sets showed high chromosome specificity in silico for the genome sequences of all 18 wheat and barley cultivars tested. The MPCR primers proved experimentally also chromosome-specific for the reference cultivars as well as for 13 additional wheat and four barley genotypes. Analyses of 16 wheat × barley F1 hybrid plants demonstrated that the MPCR primer sets enable the fast and one-step detection of all wheat and barley chromosomes. Finally, the established genotyping system was fully corroborated with the standard genomic in situ hybridisation (GISH) technique.ConclusionsWheat and barley chromosome-specific MPCR offers a fast, labour-friendly, and versatile alternative to molecular cytogenetic detection of individual chromosomes. This method is also suitable for the high-throughput analysis of distinct (sub)genomes, and, in contrast to GISH, can be performed with any tissue type. The designed primer sets proved to be highly chromosome-specific over a wide range of wheat and barley genotypes as well as in wheat × barley hybrids. The described primer design strategy can be extended to many species with precise genome sequence information.