Blood Cancer Discovery最新文献

{"title":"Abstract A18: STC-15, a novel METTL3 inhibitor, and its combination with Venetoclax confer anti-tumour activity in AML models","authors":"L. Vasiliauskaitė, Y. Ofir-Rosenfeld, M. Albertella, C. Hoareau-Aveilla, Jerry McMahon, Oliver Rausch","doi":"10.1158/2643-3249.aml23-a18","DOIUrl":"https://doi.org/10.1158/2643-3249.aml23-a18","url":null,"abstract":"\u0000 N6-methyladenosine (m6A) is one of the most abundant RNA modifications, which influences mRNA and lncRNA localization, half-life, translation, and splicing. The majority of m6A modifications on cellular mRNAs are deposited by the RNA methyltransferase METTL3. To date, METTL3 has been implicated in the initiation and progression of multiple cancer types, with the highest expression of METTL3 mRNA observed in acute myeloid leukemia (AML). Currently, one line of standard of care therapy for AML patients is Venetoclax, which targets the anti-apoptotic protein BCL2. It was shown that m6A, deposited by METTL3 on BCL2 transcript, affects BCL2 mRNA stability and translation. Storm Therapeutics has developed potent and selective METTL3 inhibitors, including the clinical candidate STC-15. Here, we explore pharmacological inhibition of METTL3 as monotherapy or in combination with Venetoclax in AML models in vitro and in vivo. Sulforhodamine B and CellTiterGloTM assays were used to assess the viability of AML cell lines and patient-derived xenografts (PDXs), respectively, following METTL3 inhibition in vitro. BCL2 protein level was evaluated by Western blotting. SynergyFinder software was used to assess the degree of synergy between METTL3 inhibitors and Venetoclax. Intra-tibial implantation of human-derived AML cells (AML-PDXs) in NSG mice was used to determine single agent and combination therapy efficacy. Multiple AML cells lines and AML-PDXs were sensitive to pharmacological inhibition of METTL3 in vitro, as assessed by loss of viability. Treatment with METTL3 inhibitors led to downregulation of BCL2 protein level in several AML cell lines, as previously suggested by literature. Based on these results, the synergy between METTL3 inhibition and Venetoclax was assessed. Matrix-combination experiments have shown a high degree of synergy between the two drugs (defined by a synergy score >10) in THP-1 and MOLM-13 cell lines. To test METTL3 inhibition as a monotherapy and in combination with Venetoclax in vivo, three AML-PDX studies were initiated. Significantly lower spleen weight was observed in all animals treated with STC-15 or STC-15 + Venetoclax, and reduced number of circulating hCD45+ cells was observed in 2 out of the 3 models. In one of the models, STC-15 monotherapy outperformed Venetoclax (median survival 68 days vs 58 days, respectively), while the combination therapy extended median group survival to 85 days in comparison to 51.5 days in the vehicle group. In conclusion, we demonstrated that METTL3 inhibition results in anti-tumour effects across different AML models. Moreover, we demonstrated a synergistic effect between the novel METTL3 inhibitor STC-15 and Venetoclax, both in vitro and in vivo. These studies provide evidence for the utility of METTL3 inhibitor as a new therapeutic agent to treat AML. Currently, STC-15 is under clinical development (NCT05584111).\u0000 Citation Format: Lina Vasiliauskaite, Yaara Ofir-Rosenfeld, Mark Albertella, Co","PeriodicalId":29944,"journal":{"name":"Blood Cancer Discovery","volume":null,"pages":null},"PeriodicalIF":11.2,"publicationDate":"2023-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46808001","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract A04: Modulation of RNA splicing enhances response to BCL2 inhibition in leukemia","authors":"Eric Wang, Omar Abdel-Wahab, Robert K Bradley, Jose Mario Bello, Won Jun Kim, Carine Bossard","doi":"10.1158/2643-3249.aml23-a04","DOIUrl":"https://doi.org/10.1158/2643-3249.aml23-a04","url":null,"abstract":"Abstract Therapy resistance is a major challenge in the treatment of cancer. Here, we performed CRISPR/Cas9 screens across a broad range of therapies used in acute myeloid leukemia to identify genomic determinants of drug response. Our screens uncovered a selective dependency on RNA splicing factors whose loss preferentially enhanced response to the BCL2 inhibitor venetoclax. Loss of the splicing factor RBM10 augmented response to venetoclax in leukemia yet was completely dispensable for normal hematopoiesis. Combined RBM10 and BCL2 inhibition led to mis-splicing and inactivation of the inhibitor of apoptosis XIAP and downregulation of BCL2A1, an anti-apoptotic protein implicated in venetoclax resistance. A novel inhibitor of splicing kinase families CLKs and DYRKs led to aberrant splicing of key splicing and apoptotic factors that synergized with venetoclax and overcame resistance to BCL2 inhibition. Our findings underscore the importance of splicing in modulating response to therapies and provide a strategy to improve venetoclax-based treatments. Citation Format: Eric Wang, Omar Abdel-Wahab, Robert K Bradley, Jose Mario Bello, Won Jun Kim, Carine Bossard. Modulation of RNA splicing enhances response to BCL2 inhibition in leukemia [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A04.","PeriodicalId":29944,"journal":{"name":"Blood Cancer Discovery","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"136096448","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract A07: Single-cell proteomic assessment of FLT3-ITD AML landscape identifies distinct resistance patterns","authors":"Li Li, M. Muftuoglu, Mahesh Basyal, N. Daver, M. Andreeff","doi":"10.1158/2643-3249.aml23-a07","DOIUrl":"https://doi.org/10.1158/2643-3249.aml23-a07","url":null,"abstract":"\u0000 Acute myeloid leukemia (AML) represents a heterogeneous hematopoietic disorder characterized by accumulation of immature hematopoietic precursors with differentiation block. FLT3 internal tandem duplications (FLT3-ITD) are commonly occurring genetic alterations in AML and are associated with poor prognosis. Preclinical studies showed combining FLT3 inhibitor with MDM2 inhibitor, which is a negative regulator of p53, was synergistic in FLT3-ITD/TP53 wild type (WT) AML. We performed single-cell proteomic evaluation of leukemia landscape in FLT3-ITD patients treated with FLT3i and MDM2i to assess proteomic profiles associated with response and resistance. We performed CyTOF analysis of leukemia cells in serially collected samples from six FLT3-ITD and TP53 WT AML patients following treatment with FLT3i+MDM2i treatment. Using 51 features assessed in CyTOF, we first performed UMAP dimension reduction and clustering to identify distinct cells in leukemia compartment. Notably, the frequencies of blasts identified through CyTOF data analysis were compatible with clinical lab reports. In line with previous reports, we also detected that NPM mutant AML cells did not express CD34. Of note, CD34+ and CD34- leukemia cells were clustered together in NPM1 mut patients, indicating that they have overlapping proteomic profiles. Interestingly, the CD34+ leukemia cells were eliminated at the early time points in CR patients while the CD34- leukemia cells were still detectable after two months. On the other hand, CD34+ leukemia cells in nonresponders(NR) persisted despite therapy. These findings indicate CD34+ leukemia cells were more sensitive to the treatment compared to NPM1 mutant CD34- leukemia cells. Next, we interrogated leukemia proteomic landscape in serial samples, evaluated the therapy-induced alterations in proteomic profiles and sought to identify potential adaptive mechanisms. To this end, we performed differential expression analysis and observed that signaling pathways (p-4EBP1, p-GSK3, p-MEK1/2, p-S6) and differentiation markers(CD33, CD68,CXCR4 HLADR) were more enriched on day 8 post treatment in NR patients, revealing that compensatory signaling activity, phenotypic profiles and differentiation status could be associated with therapy response. Moreover, we found that leukemia cells in NR patients had distinct phenotypic profiles(CD11b,CD68,CXCR4), higher levels of anti-apoptotic molecules (BCL2,MCL1) and enriched survival pathways (p-GSK3, YTHDF2) compared to baseline. In contrast, we did not observe rebound increases in CR patients post-therapy. These findings demonstrate high levels of BCL2 and MCL1 and preferential survival of more differentiated cells may be associated with therapy resistance and treatment failure in NR patient treated with MDM2i and FLT3i. In conclusion, multiplexed single-cell proteomic analysis permitted longitudinal monitoring of leukemia landscape and identified proteomic alterations associated with therapy resistance.\u0000","PeriodicalId":29944,"journal":{"name":"Blood Cancer Discovery","volume":null,"pages":null},"PeriodicalIF":11.2,"publicationDate":"2023-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"43229682","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract IA09: Epigenomic landscapes of MDS","authors":"M. Figueroa","doi":"10.1158/2643-3249.aml23-ia09","DOIUrl":"https://doi.org/10.1158/2643-3249.aml23-ia09","url":null,"abstract":"\u0000 While significant progress has been made to understand the genetic landscape of MDS, less is known about the epigenetic makeup of this disease and how this may impact biology and response to azacitidine (AZA). To address this, we performed genomic, epigenomic and transcriptomic analysis on CD34+ cells from a multicenter cohort of 94 intermediate or higher risk MDS patients treated with AZA who had documented responses. DNA methylation (DNAme) by ERRBS, gene expression (GE) by RNA-seq, mutational profiling, and detailed clinical, cytogenetic and laboratory data were documented. Supervised and unsupervised analyses revealed that aberrant DNAme in MDS is not distributed randomly but rather is highly correlated with disease phenotypes, capturing clinically relevant heterogeneity, beyond what is identified by methodologies used in the past that focused solely on promoter regions. Moreover, this epigenetic information can be harnessed for the development of robust biomarkers predictive of AZA response and integrative approaches combining GE and DNAme data can further improve the predictive performance of these biomarkers, with an AUC score=0.92.\u0000 Citation Format: Maria E. Figueroa. Epigenomic landscapes of MDS [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr IA09.","PeriodicalId":29944,"journal":{"name":"Blood Cancer Discovery","volume":null,"pages":null},"PeriodicalIF":11.2,"publicationDate":"2023-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"45018240","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract A16: Multi-omic analyses in genetically engineered mice reveal distinct and opposite effects of leukemogenic Idh and Tet2 mutations in hematopoietic stem and progenitor cells","authors":"J. Fortin, Ming-Feng Chiang, Cem Meydan, J. Foox, P. Ramachandran, Julie Leca, F. Lemonnier, Wanda Li, Miki S Gams, T. Sakamoto, Mandy F. Chu, Chantal Tobin, Eric Laugesen, Troy M Robinson, A. You-Ten, D. Butler, T. Berger, M. Minden, R. Levine, C. Guidos, A. Melnick, C. Mason, T. Mak","doi":"10.1158/2643-3249.aml23-a16","DOIUrl":"https://doi.org/10.1158/2643-3249.aml23-a16","url":null,"abstract":"\u0000 Mutations in IDH1, IDH2, and TET2 are recurrently observed in myeloid neoplasms. IDH1 and IDH2 encode isocitrate dehydrogenase isoforms, which normally catalyze the conversion of isocitrate to α-ketoglutarate (α-KG). Oncogenic IDH1/2 mutations confer neomorphic activity, leading to the production of D-2-hydroxyglutarate (D-2-HG), a potent inhibitor of α-KG-dependent enzymes which include the TET methylcytosine dioxygenases. Given their mutual exclusivity in myeloid neoplasms, IDH1, IDH2, and TET2 mutations may converge on a common oncogenic mechanism. Contrary to this expectation, we observed that they have distinct, and even opposite, effects on hematopoietic stem and progenitor cells in genetically engineered mice. Endogenous Idh2R172K caused much higher D-2-HG production compared to Idh1R132H and Idh2R140Q. This led to profound alterations in hematopoietic progenitor differentiation and to the development of myelodysplastic syndrome-like disease, with shorter survival compared to Tet2−/- mice. Epigenetic and single-cell transcriptomic analyses revealed that Idh2R172K and Tet2 loss-of-function have divergent and opposite effects on the expression and activity of key hematopoietic and leukemogenic regulators. Notably, chromatin accessibility and transcriptional deregulation in Idh2R172K cells were partially disconnected from DNA methylation alterations. These results highlight unanticipated divergent effects of IDH1/2 and TET2 mutations, which may inform the development of genotype-specific therapies.\u0000 Citation Format: Jerome Fortin, Ming-Feng Chiang, Cem Meydan, Jonathan Foox, Parameswaran Ramachandran, Julie Leca, Francois Lemonnier, Wanda Y. Li, Miki S. Gams, Takashi Sakamoto, Mandy Chu, Chantal Tobin, Eric Laugesen, Troy M. Robinson, Annick You-Ten, Daniel J. Butler, Thorsten Berger, Mark D. Minden, Ross L. Levine, Cynthia J. Guidos, Ari M. Melnick, Christopher E. Mason, Tak W. Mak. Multi-omic analyses in genetically engineered mice reveal distinct and opposite effects of leukemogenic Idh and Tet2 mutations in hematopoietic stem and progenitor cells [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A16.","PeriodicalId":29944,"journal":{"name":"Blood Cancer Discovery","volume":null,"pages":null},"PeriodicalIF":11.2,"publicationDate":"2023-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46359507","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"In Vivo Screening Unveils Pervasive RNA-Binding Protein Dependencies in Leukemic Stem Cells and Identifies ELAVL1 as a Therapeutic Target.","authors":"Ana Vujovic,&nbsp;Laura de Rooij,&nbsp;Ava Keyvani Chahi,&nbsp;He Tian Chen,&nbsp;Brian A Yee,&nbsp;Sampath K Loganathan,&nbsp;Lina Liu,&nbsp;Derek C H Chan,&nbsp;Amanda Tajik,&nbsp;Emily Tsao,&nbsp;Steven Moreira,&nbsp;Pratik Joshi,&nbsp;Joshua Xu,&nbsp;Nicholas Wong,&nbsp;Zaldy Balde,&nbsp;Soheil Jahangiri,&nbsp;Sasan Zandi,&nbsp;Stefan Aigner,&nbsp;John E Dick,&nbsp;Mark D Minden,&nbsp;Daniel Schramek,&nbsp;Gene W Yeo,&nbsp;Kristin J Hope","doi":"10.1158/2643-3230.BCD-22-0086","DOIUrl":"10.1158/2643-3230.BCD-22-0086","url":null,"abstract":"<p><p>Acute myeloid leukemia (AML) is fueled by leukemic stem cells (LSC) whose determinants are challenging to discern from hematopoietic stem cells (HSC) or uncover by approaches focused on general cell properties. We have identified a set of RNA-binding proteins (RBP) selectively enriched in human AML LSCs. Using an in vivo two-step CRISPR-Cas9 screen to assay stem cell functionality, we found 32 RBPs essential for LSCs in MLL-AF9;NrasG12D AML. Loss-of-function approaches targeting key hit RBP ELAVL1 compromised LSC-driven in vivo leukemic reconstitution, and selectively depleted primitive malignant versus healthy cells. Integrative multiomics revealed differentiation, splicing, and mitochondrial metabolism as key features defining the leukemic ELAVL1-mRNA interactome with mitochondrial import protein, TOMM34, being a direct ELAVL1-stabilized target whose repression impairs AML propagation. Altogether, using a stem cell-adapted in vivo CRISPR screen, this work demonstrates pervasive reliance on RBPs as regulators of LSCs and highlights their potential as therapeutic targets in AML.</p><p><strong>Significance: </strong>LSC-targeted therapies remain a significant unmet need in AML. We developed a stem-cell-adapted in vivo CRISPR screen to identify key LSC drivers. We uncover widespread RNA-binding protein dependencies in LSCs, including ELAVL1, which we identify as a novel therapeutic vulnerability through its regulation of mitochondrial metabolism. This article is highlighted in the In This Issue feature, p. 171.</p>","PeriodicalId":29944,"journal":{"name":"Blood Cancer Discovery","volume":null,"pages":null},"PeriodicalIF":11.2,"publicationDate":"2023-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10150294/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9682577","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract A30: Activation of GCN2 by HC-7366 results in significant anti-tumor efficacy as monotherapy and in combination with Venetoclax in AML models","authors":"Feven Tameire, P. Wojnarowicz, S. Fujisawa, Sharon Huang, O. Reilly, C. Dudgeon, Nick Collette, J. Drees, Kathryn T. Bieging-Rolett, Takashi O. Kangas, Weiyu Zhang, M. Fumagalli, Iman Dewji, Yunfang Li, Anissa S. H. Chan, Xiaohong X. Qiu, B. Harrison, Ashley LaCayo, K. Staschke, A. C. Rigby, S. Ramurthy, E. Lightcap, D. Surguladze, N. Bose","doi":"10.1158/2643-3249.aml23-a30","DOIUrl":"https://doi.org/10.1158/2643-3249.aml23-a30","url":null,"abstract":"\u0000 The integrated stress response (ISR) is an adaptive signaling pathway that cells utilize to respond to a wide range of extrinsic and intrinsic stresses, many of which are important for tumorigenesis. Activation of ISR plays a dual role in cell fate decisions; while the ISR promotes survival, prolonged activation of ISR induces apoptosis. Activation of General Control Nonderepressible 2 (GCN2), an ISR kinase that senses and responds to nutrient stress conditions results in anti-tumor activity. We are developing HC-7366, a first-in-class, first-in-human GCN2 activator, and are currently evaluating it in a phase 1 clinical trial in solid tumors (NCT05121948). Here we present a series of studies characterizing the antitumor effects of HC-7366 in acute myeloid leukemia (AML). Higher expression of GCN2 and ISR markers such as ATF4 has been observed in primitive or minimally differentiated AML cells, suggesting that AML may be particularly sensitive to HC-7366. Encouragingly, in vivo efficacy studies in MOLM-16 and KG-1 tumor models showed 100% complete response and 100% tumor growth inhibition, respectively. Analysis of tumors from treated mice by IHC demonstrated activation of ISR as evidenced by increased expression of the ATF4 targets, ASNS and PSAT1, confirming that HC-7366 is functioning as a GCN2 activator in vivo. In the MV4-11 model, a differentiated subtype of AML that shows limited response to venetoclax, the combination of HC-7366 and venetoclax produced strong benefit resulting in 26% tumor regression. Enhanced activation of ISR pathway was again observed when HC-7366 was combined with venetoclax. HC-7366 treatment also impacted possible venetoclax resistance mechanisms by increasing the levels of PUMA while reducing levels of S100A8/A9 proteins. To investigate the effects of the compound on primary AML, we performed an ex vivo screen using cells from AML patients and treatment with HC-7366 showed a remarkable decrease in cell proliferation. Furthermore, in a xenotransplantable model of patient-derived AML, we found that HC-7366 significantly reduced mature myeloid (CD33+) AML cells in the bone marrow as compared to standard of care (SOC) agents, including venetoclax. We confirmed that activation of ISR, reduction in cell viability, and inhibition of protein synthesis following treatment with HC-7366 was dependent on GCN2 using CRISPR knockout cells. In addition, HC-7366 reduced mitochondrial respiration in MOLM-16 cells, suggesting effects on cellular bioenergetics. Metabolomics analyses of AML xenograft tumors showed that HC-7366 significantly altered metabolites associated with amino acid metabolism, urea cycle, and oxidative stress. Together, our in vitro and in vivo results demonstrate that HC-7366 is a potent GCN2 activator with strong antitumor activity in AML as a single agent and in combination with venetoclax, supporting its investigation in clinical trials in patients with AML.\u0000 Citation Format: Feven Tameire, Paulina M. Wojnar","PeriodicalId":29944,"journal":{"name":"Blood Cancer Discovery","volume":null,"pages":null},"PeriodicalIF":11.2,"publicationDate":"2023-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"47780604","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract A51: Divergent effects of MDS predisposing SAMD9L point mutations, in humans, mice, and cellular models","authors":"S. Sahoo, C. Goodings, S. Pruett-Miller, M. Lillo, Lei Han, Baranda S Hansen, T. Chang, T. Lammens, M. Hofmans, Marta Derecka, B. De Moerloose, M. Wlodarski","doi":"10.1158/2643-3249.aml23-a51","DOIUrl":"https://doi.org/10.1158/2643-3249.aml23-a51","url":null,"abstract":"\u0000 We previously showed that germline SAMD9 and SAMD9L (SAMD9/9L) disorders are the most common predisposition to childhood myelodysplastic syndromes with bone marrow failure (BMF). Heterozygous mutations in both genes are growth inhibitory and undergo negative selection by acquiring compensatory rescue events in blood. Here, we describe the first report of SAMD9L genetic rescue occurring during embryonal development and resulting in germline triple-allelic mosaicism (1 wildtype (WT) and 2 mutant alleles), where only one mutant allele was transmitted to each of the two diseased children. In a family of 2 affected siblings with hypocellular BMF, the Index case carried germline heterozygous SAMD9L V1512M mutation and her brother was heterozygous for V1512L mutation. The clinical presentation for Index with V1512M was more severe compared to her brother with V1512L mutation. Genetic assessment of the parents showed the asymptomatic mother to be a triple-allelic mosaic, carrying WT SAMD9L allele at ~50% frequency in all tissues, while both V1512M and V1512L mutant alleles “competed” in their allelic distribution depending on the tissue origin. Single-cell DNA sequencing on her peripheral blood (PB) revealed 3 independent diploid clones: V1512M in 14%, V1512L in 68%, and WT (due to revertant UPD7q) in 18% of cells. Because her parents were SAMD9L WT, one of the mutations likely arose de novo and underwent failed embryonic rescue attempt leading to a second mutation. Towards studying the effect on hematopoiesis in vitro, we knocked in V1512M and V1512L mutations in inducible pluripotent stem cells (iPSC). For in vivo functional validation, we created constitutive mouse models with ortholog mutations (V1507M and V1507L). Mutant hematopoietic progenitor cells from iPSC had severely compromised proliferative capacity and yielded fewer erythroid and myeloid cells. This effect was more severe in V1512M vs. V1512L mutants. The divergent mutational phenotypes were also replicated in our mouse models: two-thirds (13/19) of the heterozygous V1507M (V1507Mhet) pups died before 4 weeks of age. In contrast, V1507Lhet mice had overall survival equal to WT mice, while one-third of homozygous V1507L (V1507Lhom) mice showed decreased survival post 19 weeks of age. At baseline, V1507Mhet mice showed severe growth retardation with multisystemic issues, which were absent in the V1507L mutant models. PB cytopenia (anemia, B-cell lymphopenia) though observed in both V1507Mhet and V1507Lhom mice, the degree of severity was high in the V1507Mhet than in the V1507Lhom model. Meanwhile, V1507Lhet mice had blood counts similar to WT. In summary, V1512M compared to V1512L mutation resulted in a more severe phenotype in all analyzed model systems. This observation showed how different mutational permutations of the same amino acid can exert divergent phenotypic effects in SAMD9L BMF disorder. Therefore, together with our genetic analysis, we could postulate that the V1512L mutatio","PeriodicalId":29944,"journal":{"name":"Blood Cancer Discovery","volume":null,"pages":null},"PeriodicalIF":11.2,"publicationDate":"2023-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"44706951","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract A45: UBTF-TD molecular alterations drive leukemogenesis in pediatric AML by directly interacting and regulating the HOXB gene locus","authors":"Juan M. Barajas, Masayuki Umeda, Ryan Hiltenbrand, Reethu Krishnan, Tamara Westover, Michael P. Walsh, Jing Ma, Sherif Abdelhamed, J. Klco","doi":"10.1158/2643-3249.aml23-a45","DOIUrl":"https://doi.org/10.1158/2643-3249.aml23-a45","url":null,"abstract":"\u0000 Pediatric acute myeloid leukemia (AML) is often refractory to chemotherapy and remains a devastating disease with poor outcomes. Consequently, there is an urgent need to characterize molecular drivers of pediatric AML with the goal of developing more effective molecular therapies. Our group recently identified somatic tandem duplications (TD) in exon 13 of upstream binding transcription factor (UBTF) as a subtype-defining genomic alteration in pediatric AML that is associated with poor outcomes, high relapse rate, and measurable residual disease (MRD) positivity. However, how these alterations drive leukemogenesis is yet to be investigated. Wild-type UBTF regulates rRNA transcription, ribosome biogenesis, and nucleolar formation. Our recent work suggests that UBTF-TD retains these functions. However, we found that expression of UBTF-TD in cord blood CD34+ cells, but not wild-type UBTF, leads to their increased proliferation, self-renewal, and CD117/CD33 surface marker expression, suggesting a potential gain of function as well. Furthermore, we also observed increased expression of HOXB locus genes, PRDM16, MEIS1, and NKX2-3 in the CD34+ UBTF-TD overexpression model relative to our WT control. These expression patterns are similar to what we see in patients, collectively implicating UBTF-TD driving leukemic phenotypes. Based on our localization studies of UBTF-TD to DNA, we hypothesized that UBTF-TD protein may gain the ability to interact with genomic loci that characterize UBTF-TD AML, like the HOXB locus. We therefore wanted to test if UBTF-TD localized to the HOXB cluster and if this lead direct transcriptional upregulation. To test this, we expressed HA-tagged UBTF-TD in cord-blood CD34+ cells and mapped UBTF-TD genomic occupancy using CUT&RUN. We found that UBTF-TD localized to the promoters and gene bodies of key genes, including PRDM16, MEIS1, NKX2-3, and the HOXB cluster. These sites were accompanied by H3K4me3 and H3K27ac active marks without the H3K27me3 repressive mark. To validate these as bone-fide UBTF-TD-occupied regions, we utilized a expressed FKBP12F36V-HA-UBTF-TD fusion in CD34+ cells. This degradation tag (dTAG) system allows for rapid degradation of UBTF-TD protein upon dTAG-13 treatment. We treated our FKBP12F36V-HA-UBTF-TD expressing cord blood CD34+ cells with 1uM of dTAG-13 or DMSO. We found that a 3-day treatment with dTAG-13 led to downregulation of HOXB gene locus expression, but loss of CD117 expression or cell growth required prolonged treatment. Furthermore, our CUT&RUN data showed that dTAG-13 treatment resulted in loss of UBTF-TD protein at the HOXB gene cluster. Collectively, these data show that UBTF-TD interacts with genomic loci at the HOXB gene cluster to directly upregulate their expression. This loss of HOX gene expression leads then leads to loss of a leukemic phenotype. Our data highlights a potential mechanism for UBTF-TD leukemogenesis and sets a foundation where inhibiting the UBTF-TD molecular signa","PeriodicalId":29944,"journal":{"name":"Blood Cancer Discovery","volume":null,"pages":null},"PeriodicalIF":11.2,"publicationDate":"2023-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"45460560","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract A20: Inflammation remodels the immune microenvironment in acute myeloid leukemia","authors":"Audrey Lasry, B. Nadorp, M. Fornerod, D. Nicolet, Huiyun Wu, C. Walker, Zhengxi Sun, Matthew T. Witkowski, Anastasia N. Tikhonova, Maria Guillamot, Geraldine Cayanan, Anna H. Yeaton, T. Gruber, Ann-Kathrin Eisfeld, I. Aifantis","doi":"10.1158/2643-3249.aml23-a20","DOIUrl":"https://doi.org/10.1158/2643-3249.aml23-a20","url":null,"abstract":"\u0000 Acute myeloid leukemia (AML) is a hematopoietic malignancy with poor prognosis and limited treatment options. Here we provide a comprehensive single cell RNA-Sequencing census of the bone marrow immune microenvironment in adult and pediatric AML patients. We characterize unique inflammation signatures in a subset of AML patients, associated with inferior outcomes. We identify atypical B cells, a dysfunctional B cell subtype enriched in high-inflammation AML patients, as well as an increase in CD8+ GZMK+ and regulatory T cells, accompanied by a reduction in T cell clonal expansion. We derive an inflammation-associated gene score (iScore) that associates with poor survival outcomes in AML patients. Addition of the iScore refines current risk stratifications for AML patients and may enable identification of patients in need of more aggressive treatment. This work provides a first framework for classifying AML patients based on their immune microenvironment and a rationale for consideration of the inflammatory state in clinical settings.\u0000 Citation Format: Audrey Lasry, Bettina Nadorp, Maarten Fornerod, Deedra Nicolet, Huiyun Wu, Christopher J Walker, Zhengxi Sun, Matthew T Witkowski, Anastasia N Tikhonova, Maria Guillamot, Geraldine Cayanan, Anna Yeaton, Tanja A Gruber, Ann-Kathrin Eisfeld, Iannis Aifantis. Inflammation remodels the immune microenvironment in acute myeloid leukemia [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A20.","PeriodicalId":29944,"journal":{"name":"Blood Cancer Discovery","volume":null,"pages":null},"PeriodicalIF":11.2,"publicationDate":"2023-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46672268","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}