TPS7075
Background: Epigenetic dysregulation is a hallmark of acute myeloid leukemia (AML) and is caused by recurrent translocations and/or mutations in chromatin regulators and transcription ...factors, resulting in myeloid differentiation blockade and leukemic stem cell renewal. Accordingly, about 70% of recurring mutations in AML patients (pts) target regulators of gene expression, underscoring the potential of epigenetic therapies to change the disease natural history. Iadademstat (iada/ORY-1001) is a specific, oral, potent, covalent inhibitor of the epigenetic Lysine-Specific Demethylase 1 (LSD1/KDMA1) enzyme. In preclinical and clinical studies iada decreased leukemic stem cell survival and induced macrophage/monocytic differentiation of blasts. ALICE, a Ph2 study of iada in combination with azacitidine (aza), showed high complete remission (CR/CRi) rates and durable responses in treatment naïve, unfit AML pts without exacerbating the toxicity profile of aza (Salamero et al., Oral at ASH 2022). Despite improvements in AML therapy, relapsed and refractory (R/R) cases are frequent and contribute to the death of more than 50% of pts, particularly in those subpopulations with higher risk genetics, the largest of which (up to 30-40% AML pts) harbors fms-like tyrosine kinase 3 mutations (FLT3mut+). Use of the FLT3 inhibitor (FLT3i) gilteritinib as monotherapy for R/R pts resulted in improved outcomes but the duration of remission achieved is transient and often brief (CR rate: 20%; EFS: 2.8 months) per the ADMIRAL Ph3 study (Perl, et al., NEJM 2019). Preclinically, iada has marked synergy with FLT3i, particularly gilteritinib, in FLT3 wild-type and FLT3 mut+ AML cells and in derived cell lines resistant to venetoclax, azacitidine and FLT3is. Methods: Adult pts with ECOG 0-2 and FLT3 mut+ R/R AML, after 1 or 2 prior lines of therapy, will be enrolled. In escalation (3+3), up to 18 pts will receive iada at 75 to 150 ug, orally, in 5 days ON-2 days OFF schedule, with gilteritinib SoC. Up to approximate 14 pts will be expanded from pharmacologically active dose/s (per Project OPTIMUS). Primary endpoints of the study are safety and RP2D determination. Bayesian posterior probability efficacy monitoring will be performed periodically for each dose cohort. Bayesian efficacy futility and early stopping boundary will be applied. Posterior probability criterion (Prob (CR > 0.3) ≥ 0.60) at the end of the study will warrant further research. Safety in expansion will be evaluated continuously with a Bayesian stopping rule. Secondary endpoints include OS, EFS, CR, CR/CRh, ORR, TTR, DoR, and transfusion rate. Exploratory endpoints include PK, PD, MRD and gene mutational analysis. The study will run in 15 US sites. Additional sites will be added for a subsequent Phase 2 randomized controlled double-blinded FRIDA 2 study to assess the efficacy of this combination in R/R FLT3 mut+ AML. Clinical trial information: NCT05546580 .
Correct vascular differentiation requires distinct patterns of gene expression in different subtypes of endothelial cells. Members of the ETS transcription factor family are essential for the ...transcriptional activation of arterial and angiogenesis-specific gene regulatory elements, leading to the hypothesis that they play lineage-defining roles in arterial and angiogenic differentiation directly downstream of VEGFA signalling. However, an alternative explanation is that ETS binding at enhancers and promoters is a general requirement for activation of many endothelial genes regardless of expression pattern, with subtype-specificity provided by additional factors. Here we use analysis of Ephb4 and Coup-TFII (Nr2f2) vein-specific enhancers to demonstrate that ETS factors are equally essential for vein, arterial and angiogenic-specific enhancer activity patterns. Further, we show that ETS factor binding at these vein-specific enhancers is enriched by VEGFA signalling, similar to that seen at arterial and angiogenic enhancers. However, while arterial and angiogenic enhancers can be activated by VEGFA in vivo, the Ephb4 and Coup-TFII venous enhancers are not, suggesting that the specificity of VEGFA-induced arterial and angiogenic enhancer activity occurs via non-ETS transcription factors. These results support a model in which ETS factors are not the primary regulators of specific patterns of gene expression in different endothelial subtypes.
•Vein-specific enhancers can contain essential ETS motifs.•VEGFA induced an increase in ETS binding at vein, arterial and angiogenic enhancers.•VEGFA stimulation cannot induce vein-specific enhancer activity.
Abstract only
e14248
Background: By recruiting the immune system against cancer cells, immunotherapy holds promise to achieve long-lasting responses in several malignant conditions. However, a ...therapeutic response is observed only in a subset of patients. The low mutational burden of “cold” tumors enables them to stay undetected from the host inflammatory response (innate resistance). Also, cancer cells develop a number of coping strategies in response to the selective pressure applied by the treatment with checkpoint inhibitors (recruitment of regulatory cells, defective antigen presentation, immunosuppressant mediators, reduced costimulation, and T cell apoptosis). Hence combinatorial treatments are being considered to tackle the problem of resistances and responsiveness. Iadademstat (ORY-1001) is a selective, orally-available KDM1A inhibitor with low nanomolar potency (Maes et al., 2018). KDM1A is a histone demethylase involved in transcription regulation and key to tumorigenicity in different hematological and solid malignancies. The tolerability and safety of iadademstat has been evaluated in Phase I studies in AML and SCLC and the drug is currently being investigated in combination with SoC in phase II clinical trials in AML and in SCLC. Methods: The combined efficacy of iadademstat administered orally at 10 µg/kg, a dose that does not provoke weight loss or thrombocytopenia, and an anti-Pd1 antibody was evaluated in the mice syngeneic B16F10 melanoma model. Results: Treatment with iadademstat and the checkpoint inhibitor anti-Pd1 monoclonal antibody was well tolerated. By Day 15, treatment with the anti-Pd1 antibody alone or in combo with iadademstat reduced tumor growth by 45% and 65% relative to vehicle controls. Control animals were sacrificed by day 19 due to progression. By day 22, 54% reduction was achieved in the animals treated with the combination compared to the animals treated with the anti-Pd1 antibody alone. Conclusions: This proof-of-concept in vivo experiment highlights the potential of iadademstat in combination with checkpoint inhibitors, prompting further investigation in other oncological indications, characterized by low or no response to the currently approved immune-oncology treatments.
Abstract
Background: Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive and rare malignancies accounting for 10% of all tissue sarcomas. Around 50% develop in patients with ...neurofibromatosis type 1 carrying mutations in the NF1 gene and the other 50% occur sporadically. NF1 is a tumor suppressor gene and a negative regulator of RAS signaling pathway. However, mutation of the NF1 gene is not sufficient to drive MPNST and a series of further mutational events must accumulate in genes other than NF1, such as in CDKN2A and PRC2 complex components. Mutations in p53 or PTEN and/or mutations or copy number variations in oncogenes, such as EGFR or c-Met can also be detected in MPNST. Herein we explore the effects of LSD1 inhibition with iadademstat as a single agent in a panel of MPNST cell lines, both NF1-related and sporadic, and in combination with inhibitors of the MEK-ERK and PI3K/AKT signaling pathways.
Materials and Methods: The cell viability in 12 MPNST cell lines, both commercial and derived from patients’ primary tumors, was assessed after 6 days of treatment with iadademstat as a single agent. Synergisms between iadademstat and the MEK inhibitor selumetinib or the PI3K inhibitor copanlisib were also evaluated after 6 days of treatment in three selected responsive cell lines and analyzed with Calcusyn and Combenefit software. Additionally, the effects of iadademstat in RAS pathways (pAKT and pERK levels) were also evaluated in these cell lines.
Results: Iadademstat displayed sub-nanomolar activity as a single agent in 8 out of 12 cell lines tested, both NF1-related and sporadic, with different degrees of viability reduction. This was further confirmed with two additional LSD1 inhibitors, with iadademstat being the most potent inhibitor tested. Highly-responsive cell lines showed a decrease in pAKT levels while medium- and low-responsive cell lines displayed no significant differences. pERK levels were not affected. Moreover, the combination of iadademstat and selumetinib or copanlisib displayed strong synergy in cell viability assays in both commercial and patient-derived iadademstat-sensitive cell lines.
Conclusions: The LSD1 inhibitor iadademstat is efficacious as single agent in nearly 70% of the MPNST cell lines tested, both NF1-related and sporadic and this efficacy increases synergistically in combination with selumetinib or copanlisib. Therefore, LSD1 inhibitors alone or in combination emerge as a potential treatment for MPNST, an indication with high unmet needs with no approved targeted therapies available.
Citation Format: Natalia Sacilotto, Cristina Mascaró, Edgar Creus, Juana Fernández, Conxi Lázaro, Ana Limón, Robert Soliva, Jordi Xaus. The LSD1 inhibitor iadademstat shows preclinical efficacy in malignant peripheral nerve sheath tumor cells and synergistic effects in combination 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 A061.
The ability of p53 to regulate transcription is crucial for tumor suppression and implies that inherited polymorphisms in functional p53-binding sites could influence cancer. Here, we identify a ...polymorphic p53 responsive element and demonstrate its influence on cancer risk using genome-wide data sets of cancer susceptibility loci, genetic variation, p53 occupancy, and p53-binding sites. We uncover a single-nucleotide polymorphism (SNP) in a functional p53-binding site and establish its influence on the ability of p53 to bind to and regulate transcription of the KITLG gene. The SNP resides in KITLG and associates with one of the largest risks identified among cancer genome-wide association studies. We establish that the SNP has undergone positive selection throughout evolution, signifying a selective benefit, but go on to show that similar SNPs are rare in the genome due to negative selection, indicating that polymorphisms in p53-binding sites are primarily detrimental to humans.
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•A common SNP can influence p53’s regulation of a key target gene, the KITLG gene•The SNP in the p53-binding site can significantly influence human cancer risk•The SNP in the p53-binding site has undergone positive natural selection in humans•Similar SNPs in p53-binding sites are rare in the genome due to negative selection
The polymorphism in the KITLG regulatory region associates with differential testicular cancer risk (higher with the G allele, which is more prevalent in Caucasians than in African American males) but also shows signs of positive natural selection, perhaps due to the role of KIT in UV protection in light skinned Caucasians.
Abstract
Lysine Specific Demethylase 1 (LSD1, also known as KDM1A) is a member of the FAD-containing family of lysine demethylases and is generally involved in repressing transcription by ...specifically removing methyl groups from mono- and di-methylated lysine 4 of histone 3 - epigenetic marks commonly associated with actively transcribed genes. LSD1 over-expression has been associated with disease progression and worse prognosis in several human cancers, and its inhibition has been shown to reduce cancer cell growth, migration and invasion. LSD1 has therefore been recognized as a target of interest for the development of new drugs to treat cancer. Iadademstat (aka ORY-1001), a highly potent and selective LSD1 inhibitor (LSD1i) in clinical development for malignant indications, and other LSD1is, have been reported to be effective for the treatment of small cell lung cancer (SCLC) in in vitro and in vivo models. However, the published data indicate that while certain SCLC are highly sensitive to LSD1 inhibition this sensitivity is not a universal feature of SCLC cells raising the need to develop methods for identifying sensitive SCLC tumors. Herein, we report the identification of the predictive biomarkers ASCL1 and SOX2 for the enrichment of the SCLC population more likely to respond to iadademstat and other LSD1is. Response to LSD1is was evaluated in a broad panel of SCLC cell lines (from viability assay data obtained internally at Oryzon Genomics with iadademstat and from data published elsewhere using other LSD1is). Additionally, analysis of gene expression was performed in this cell line panel using internal and publicly available datasets (Affymetrix, CCLE). This analysis revealed that the transcription factors ASCL1 and SOX2 are more likely to be highly expressed in SCLC cell lines that respond to LSD1 inhibition. Moreover, the mRNA levels of these biomarkers were highly correlated with their protein levels in both SCLC cell lines and in patient-derived xenografts. In particular, immunofluorescence staining of these patient-derived SCLC samples can discriminate different degrees of ASCL1 and SOX2 protein expression levels (none, low, medium, high), which correlate with their mRNA levels determined by RNAseq in the same samples. Moreover, analysis of ASCL1 and SOX2 protein levels analyzed internally by immunofluorescence in 40 SCLC biopsies showed that 58% samples concomitantly expressed medium to high levels of both biomarkers. Taken together, these data suggest that medium to high expression of both ASCL1 and SOX2 define a SCLC population more likely to respond to LSD1 inhibition, and that this population could account for nearly 60% of SCLC cases. Therefore, ASCL1 and SOX2 expression could be used as predictive biomarkers for personalized medicine with iadademstat in SCLC patients and clinical research to validate this hypothesis is warranted.
Citation Format: Natalia Sacilotto, Serena Lunardi, Filippo Ciceri, Cristina Mascaro, Tamara Maes, Ana Limon, Douglas V. Faller. ASCL1 and SOX2 expression levels predict sensitivity to LSD1 inhibition with iadademstat in small cell lung cancer. abstract. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B025.
OBJECTIVE—The vascular endothelial growth factor (VEGF) receptor Flk1 is essential for vascular development, but the signaling and transcriptional pathways by which its expression is regulated in ...endothelial cells remain unclear. Although previous studies have identified 2 Flk1 regulatory enhancers, these are dispensable for Flk1 expression, indicating that additional enhancers contribute to Flk1 regulation in endothelial cells. In the present study, we sought to identify Flk1 enhancers contributing to expression in endothelial cells.
APPROACH AND RESULTS—A region of the 10th intron of the Flk1 gene (Flk1in10) was identified as a putative enhancer and tested in mouse and zebrafish transgenic models. This region robustly directed reporter gene expression in arterial endothelial cells. Using a combination of targeted mutagenesis of transcription factor–binding sites and gene silencing of transcription factors, we found that Gata and Ets factors are required for Flk1in10 enhancer activity in all endothelial cells. Furthermore, we showed that activity of the Flk1in10 enhancer is restricted to arteries through repression of gene expression in venous endothelial cells by the Notch pathway transcriptional regulator Rbpj.
CONCLUSIONS—This study demonstrates a novel mechanism of arterial–venous identity acquisition, indicates a direct link between the Notch and VEGF signaling pathways, and illustrates how cis-regulatory diversity permits differential expression outcomes from a limited repertoire of transcriptional regulators.
Abstract
Histone Lysine demethylases (KDMs) are emerging clinical targets in cancer. However, a characterization of the mechanism of action of KDMs small molecule inhibitors is limited by the fact ...that these targets act at multiple levels, having several substrates, different scaffolding roles and regulating gene expression in a cell-specific manner. We developed biotinylated chemoprobes capable of binding to the active site of different KDMs and demonstrated their versatility in target engagement and chemoproteomics analysis. First, we synthetized a biotinylated chemoprobe capable of binding selectively and irreversibly to the active form of KDM1A. By coupling the chemoprobe to an immune-based assay, we can quantify the levels of free KDM1A relative to total levels of KDM1A in a dose-dependent manner in vitro and in vivo. Using this assay, we measured the KDM1A target engagement in models for AML and SCLC. These data were used to perform quantitative pharmacological modeling of SCLC xenograft models treated with ORY-1001 (see Medhi et al, AACR 2018 submitted abstract). The technique can be readily applied to clinical samples and has been used to characterize the pharmacokinetic / pharmacodynamic relationship of KDM1A inhibitors in Phase I trials. In addition, we show that the proteomic profile of the chemoprobe pulldown mirrors the binding partners of inhibited KDM1A. Our proteomic analysis using the KDM1A specific chemoprobe in AML cells identified several ZNF transcription factors but SNAG domain proteins, known to bind to the inhibitor binding site, were not retrieved, providing insight in the interaction changes induced by a KDM1A inhibitor. The strategy was also applied to other targets such as Jumonji C (JmjC) domain-containing proteins like the KDM5 demethylase family, reported to play a role in the emergence of drug tolerance. Overall, this study offers important insights and tools for investigating the role of KDM inhibitors in cancer and their further development into the clinical setting.
Citation Format: Serena Lunardi, Filippo Ciceri, Cristina Mascaró Crusat, Raquel Ruiz, Elena Carceller, Alberto Ortega, Natalia Sacilotto, Paola Dessanti, Jordi Salas, Michele Lufino, Tamara Maes. Novel multi-functional chemoprobes for the characterization of the mechanism of action of histone lysine demethylases inhibitors in cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-003.
Gas1 (growth arrest-specific 1) gene is known to inhibit cell proliferation in a variety of models, but its possible implication in regulating quiescence in adult tissues has not been examined to ...date. The knowledge of how Gas1 is regulated in quiescence may contribute to understand the deregulation occurring in neoplastic diseases. Gas1 expression has been studied in quiescent murine liver and during the naturally synchronized cell proliferation after partial hepatectomy. Chromatin immunoprecipitation at nucleosomal resolution (Nuc-ChIP) has been used to carry out the study preserving the in vivo conditions. Transcription has been assessed at real time by quantifying the presence of RNA polymerase II in coding regions (RNApol-ChIP). It has been found that Gas1 is expressed not only in quiescent liver but also at the cell cycle G.sub.1 /S transition. The latter expression peak had not been previously reported. Two nucleosomes, flanking a nucleosome-free region, are positioned close to the transcription start site. Both nucleosomes slide in going from the active to the inactive state and vice versa. Nuc-ChIP analysis of the acquisition of histone epigenetic marks show distinctive features in both active states: H3K9ac and H3K4me2 are characteristic of transcription in G.sub.0 and H4R3me2 in G.sub.1 /S transition. Sequential-ChIP analysis revealed that the "repressing" mark H3K9me2 colocalize with several "activating" marks at nucleosome N-1 when Gas1 is actively transcribed suggesting a greater plasticity of epigenetic marks than proposed until now. The recruitment of chromatin-remodeling or modifying complexes also displayed distinct characteristics in quiescence and the G.sub.1 /S transition. The finding that Gas1 is transcribed at the G.sub.1 /S transition suggests that the gene may exert a novel function during cell proliferation. Transcription of this gene is modulated by specific "activating" and "repressing" epigenetic marks, and by chromatin remodeling and histone modifying complexes recruitment, at specific nucleosomes in Gas1 promoter.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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