Activation of mitogenic signaling pathways is a common oncogenic driver of many solid tumors including lung cancer. Although activating mutations in the mitogen-activated protein kinase (MAPK) ...pathway are prevalent in non-small cell lung cancers, MAPK pathway activity, counterintuitively, is relatively suppressed in the more aggressively proliferative small cell lung cancer (SCLC). Here, we elucidate the role of the MAPK pathway and how it interacts with other signaling pathways in SCLC. We find that the most common SCLC subtype, SCLC-A associated with high expression of ASCL1, is selectively sensitive to MAPK activation in vitro and in vivo through induction of cell-cycle arrest and senescence. We show strong upregulation of ERK negative feedback regulators and STAT signaling upon MAPK activation in SCLC-A lines. These findings provide insight into the complexity of signaling networks in SCLC and suggest subtype-specific mitogenic vulnerabilities.
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•MAPK activation causes cell-cycle arrest and senescence selectively in SCLC-A subtype•MAPK-induced growth inhibition is independent of NOTCH signaling•MAPK activation increases ERK negative feedback and activates STAT3 signaling
Biological sciences; Cell biology; Molecular biology
Somatic mutations in the isocitrate dehydrogenase 2 gene (IDH2) contribute to the pathogenesis of acute myeloid leukaemia (AML) through the production of the oncometabolite 2-hydroxyglutarate (2HG)
. ...Enasidenib (AG-221) is an allosteric inhibitor that binds to the IDH2 dimer interface and blocks the production of 2HG by IDH2 mutants
. In a phase I/II clinical trial, enasidenib inhibited the production of 2HG and induced clinical responses in relapsed or refractory IDH2-mutant AML
. Here we describe two patients with IDH2-mutant AML who had a clinical response to enasidenib followed by clinical resistance, disease progression, and a recurrent increase in circulating levels of 2HG. We show that therapeutic resistance is associated with the emergence of second-site IDH2 mutations in trans, such that the resistance mutations occurred in the IDH2 allele without the neomorphic R140Q mutation. The in trans mutations occurred at glutamine 316 (Q316E) and isoleucine 319 (I319M), which are at the interface where enasidenib binds to the IDH2 dimer. The expression of either of these mutant disease alleles alone did not induce the production of 2HG; however, the expression of the Q316E or I319M mutation together with the R140Q mutation in trans allowed 2HG production that was resistant to inhibition by enasidenib. Biochemical studies predicted that resistance to allosteric IDH inhibitors could also occur via IDH dimer-interface mutations in cis, which was confirmed in a patient with acquired resistance to the IDH1 inhibitor ivosidenib (AG-120). Our observations uncover a mechanism of acquired resistance to a targeted therapy and underscore the importance of 2HG production in the pathogenesis of IDH-mutant malignancies.
Abstract
Background Lung cancer is the leading cause of cancer death, killing more people than colon, breast, and prostate cancers combined (Siegel et al. 2013). Small cell lung cancer (SCLC) is a ...high grade neuroendocrine tumor accounting for ~15% of all lung cancers (Hann et al. 2019). Metastasis is often found at first diagnosis, making SCLC exceptionally lethal (2-year survival <5%) (Byers et al. 2015). Whilst MAPK mutations can be found in roughly 30% of human cancers (Schubbert et al. 2007) including non-small cell lung cancer (NSCLC), genomic and proteomic analyses have indicated suppression of MAPK pathway activity in SCLC (Cerami et al. 2012; Gao et al. 2013; Wagle et al. 2018). This striking difference is not well understood and previous attempts to determine whether this might be therapeutically important (Ravi et al, 1998; Cristea et al. 2020) have had conflicting conclusions. SCLC has recently been defined by the relative expression of four major transcriptional regulators (ASCL1, NeuroD1, POU2F3, YAP1) (Rudin et al., 2019). In this study we aimed to elucidate the effect of MAPK activation in these different SCLC subtypes and explore its therapeutic vulnerability.
Results We used a doxycycline-inducible vector for expression of MEKDDS217D/S221D (MEK1) in a cohort of ASCL1-, NEUROD1, and POU2F3- driven cell lines. Activation through MEK1 in ASCL1-driven SCLC cell lines resulted in a significant decrease in cell growth over 9 days. This was associated with a decrease in neuroendocrine markers ASCL1 and INSM1, a G2 cell cycle arrest and no significant increase in apoptotic cells. Expression of MEK1 in other SCLC subtypes and NSCLC failed to show any appreciable changes in cell growth. Remarkably, athymic mice injected with a MEK1 expressing ASCL1-driven cell line showed significantly slower tumor formation and longer survival than the ASCL1-driven cell line not expressing MEK1. Previous work established that hyperactivation of BRAFV600E, RAS and MYC can result in oncogene-induced senescence (Serrano et al. 1997) which is caused by upregulation of negative feedback such as SPRY2, DUSP6, ETV5 rather than MAPK pathway activation in some solid tumors and pre-B ALL (Courtois-Cox et al 2006; Shojaee et al. 2015). Similarly, we also observed strong upregulation of DUSP6, SPRY2, but not ETV5 upon MAPK activation. This was especially prominent in ASCL1-driven cell lines that changed from the normal phenotype of being in suspension to a more adherent morphology as a result of MAPK activation. Interestingly, phosphokinase array in the major subtype cell lines after MEK1 activation, demonstrated that, almost exclusively, the STAT pathways, in particular STAT3 through phosphorylation at S727 was strongly upregulated in the ASCL1-driven subtype. This prompted us to examine whether these cells were sensitive to STAT3 inhibition. Upon treatment with a STAT3 inhibitor, Stattic (1μM), ASCL1-driven SCLC cells reached their IC50 after 3-5 days in comparison to 9 days for other SCLC subtypes. NSCLC cells were resistant to STAT3 inhibition.
Summary These findings suggest that ASCL1-driven SCLC in vitro and in vivo is sensitive to activation of MAPK signaling in comparison to other SCLC subtypes. Whilst activation of the MAPK pathway might seem counterintuitive to current treatment strategies that aim to inhibit oncogenic signaling, we propose the use of a STAT3 inhibitor that has shown to be effective in vitro.
Citation Format: Rebecca Caeser, Christopher Hulton, Vidushi Durani, Emily Costa, Megan Little, Nisargbhai S. Shah, Elisa de Stanchina, John T. Poirier, Charles M. Rudin, Triparna Sen. MAPK pathway activation represents a therapeutic vulnerability in ASCL1-driven SCLC abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB186.
e20105 Background: Neuroendocrine (NE) transformation occurs as a mechanism of resistance to targeted therapy in up to 14% and 30% of EGFR-mutant lung and AR-dependent prostate adenocarcinomas, ...respectively, leading to poor prognosis. Even if we know the tumor population at high risk of transformation ( TP53/RB1-mutated), no therapies to prevent NE relapse are currently available. Methods: To identify therapeutic vulnerabilities for tumors undergoing NE transformation, we performed an in vitro CRISPR screen in a NE-transformed lung tumor model, followed by functional validation in in vivo prostate and lung models of NE transformation, including pharmacologic as well as genetic (isogenic cell line with overexpression or knock of CDC7) validation. Results: Our screen identified CDC7, involved in DNA replication and DNA damage response, as a potential therapeutic target in this setting. Proteogenomic analyses revealed CDC7 upregulation in lung and prostate clinical samples undergoing NE transformation, detected already in pre-transformation adenocarcinomas. Importantly, TP53/RB1-inactivation induced sensitivity to the CDC7 inhibitor simurosertib, unraveling a therapeutic vulnerability in tumors at high risk of NE transformation. Thus, we tested the combination of simurosertib with targeted therapy in vivo in different lung and prostate patient-derived models of NE transformation. In these, simurosertib was able to suppress NE transformation and dramatically delay tumor relapse. Trajectory analysis on single-cell transcriptomic data for such models revealed a NE transformation transcriptional program occurring already in the untreated tumors before transformation. CDC7 inhibition led to increased proteasomal activity and degradation of MYC, a stemness transcription factor involved in NE transformation. Ectopic overexpression of MYC T58A , a proteasome degradation-resistant MYC isoform, rescued the NE phenotype in these transformation models, suggesting that CDC7 inhibition-induced MYC degradation is the mechanism by which NE transformation is prevented. Conclusions: In sum, CDC7 inhibition may suppress, or at least dramatically delay NE transformation in patients with lung and prostate adenocarcinomas at high risk of transformation, by inducing MYC proteasomal degradation. The clinical availability of CDC7 inhibitors, currently in phase II clinical trials after demonstrating tolerability and preliminary efficacy, will allow rapid translation of these results into the clinics.
In lung and prostate adenocarcinomas, neuroendocrine (NE) transformation to an aggressive derivative resembling small cell lung cancer (SCLC) is associated with poor prognosis. We previously ...described dependency of SCLC on the nuclear transporter exportin 1. Here, we explored the role of exportin 1 in NE transformation. We observed up-regulated exportin 1 in lung and prostate pretransformation adenocarcinomas. Exportin 1 was up-regulated after genetic inactivation of TP53 and RB1 in lung and prostate adenocarcinoma cell lines, accompanied by increased sensitivity to the exportin 1 inhibitor selinexor in vitro. Exportin 1 inhibition prevented NE transformation in different TP53/RB1-inactivated prostate adenocarcinoma xenograft models that acquire NE features upon treatment with the aromatase inhibitor enzalutamide and extended response to the EGFR inhibitor osimertinib in a lung cancer transformation patient-derived xenograft (PDX) model exhibiting combined adenocarcinoma/SCLC histology. Ectopic SOX2 expression restored the enzalutamide-promoted NE phenotype on adenocarcinoma-to-NE transformation xenograft models despite selinexor treatment. Selinexor sensitized NE-transformed lung and prostate small cell carcinoma PDXs to standard cytotoxics. Together, these data nominate exportin 1 inhibition as a potential therapeutic target to constrain lineage plasticity and prevent or treat NE transformation in lung and prostate adenocarcinoma.
Abstract
Introduction Lung adenocarcinoma (LUAD) is the most common histological subtype of lung cancer, accounting for almost 50% of lung cancer cases. Comprehensive molecular characterization of ...LUAD tumors has identified actionable drivers and led to the development of targeted inhibitors that have substantially improved patient survival. Our team previously showed that concomitant mutational inactivation of Serine/Threonine Kinase 11 (STK11) and Kelch-like ECH Associated Protein1 (KEAP1), found in up to 10% of LUAD cases, enhances cell proliferation and invasion in vitro and in vivo. Bulk RNA sequencing identified upregulation of genes involved in ferroptosis, an iron-dependent form of programmed cell death, in STK11/KEAP1 double mutant models, nominating ferroptosis as a potential vulnerability in these tumors. Consistently, CRISPR/Cas9 based genetic screening identified stearoyl-CoA desaturase (SCD), a gene involved in ferroptosis protection, as a therapeutic target in the STK11/KEAP1 double mutant tumors. However, the mechanism of ferroptosis evasion in this subset in not well understood.
Methods To further characterize the role of ferroptosis regulators in STK11/KEAP1 co-mutant setting, we performed phospho-kinase arrays and RNA sequencing in STK11/KEAP1 cell lines followed by validation through western blotting. We also performed gene expression analysis in patient derived xenografts (PDX) models treated with the SCD inhibitor to further characterize potential mechanisms by which SCD inhibition has synthetic lethal effects in STK11/KEAP1 co-mutant tumors.
Results We demonstrate that SCD overexpression protects STK11/KEAP1 co-mutant LUADs from undergoing ferroptosis. Pharmacological inhibition of SCD significantly reduced viability of STK11/KEAP1 co-mutant LUADs and made the co-mutant cells sensitive to ferroptosis induction. Phospho-kinase arrays revealed decreased activation of the JAK-STAT and AKT signaling pathways in STK11/KEAP1-double KO LUADs models as compared to either STK11 or KEAP1 single mutant isogenic conditions, which was confirmed by RNA sequencing data showing downregulation of genes involved in these pathways specifically in the double knockout setting. Interestingly, SCD pharmacological inhibition by CVT-11127 reversed this phenotype and induced overexpression of genes involved in both pathways. Additionally, SCD genetic knock out in STK11/KEAP1-double KO LUADs models mimicked the effects observed after SCD pharmacological inhibition, supporting that these were derived from on-target drug action.
Conclusions To summarize, these results suggest a potential interplay between STK11/KEAP1 function loss, ferroptosis protection, and the JAK-STAT and AKT oncogenic signaling pathways in LUAD. Further study of the role of these signaling pathways in ferroptosis may reveal mechanistic insight into the aggressive nature of these tumors.
Citation Format: Vidushi Durani, Corrin A. Wohlhieter, Alvaro Quintanal-Villalonga, Triparna Sen, Parvathy Manoj, Charles M. Rudin. Ferroptosis evasion as a therapeutic strategy in STK11/KEAP1 co-mutant lung adenocarcinoma abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3000.
Abstract Introduction Small Cell Lung Cancer (SCLC) can be classified into transcriptional subtypes with distinct degrees of neuroendocrine (NE) differentiation. Recent evidence supports plasticity ...among subtypes with a bias toward adoption of low-NE states during disease progression or upon acquired chemotherapy resistance. Here, we identify a role for SMARCA4, the catalytic subunit of the SWI/SNF complex, as a regulator of subtype shift in SCLC. Methods ATACseq and RNAseq experiments were performed in SCLC cells after pharmacological inhibition of SMARCA4. DNA binding of SMARCA4 was characterized by ChIPseq in high-NE SCLC patient derived xenografts (PDXs). Enrichment analyses were applied to transcriptomic data. Combination of FHD-286 and afatinib was tested in vitro and in a set of chemo-resistant SCLC PDXs in vivo. Results SMARCA4 expression positively correlates with that of NE genes in both SCLC cell lines and patient tumors. Pharmacological inhibition of SMARCA4 with FHD-286 induces the loss of NE features and downregulates neuroendocrine and neuronal signaling pathways while activating non-NE factors. SMARCA4 binds to gene loci encoding NE-lineage transcription factors ASCL1 and NEUROD1 and alters chromatin accessibility, enhancing NE programs. Enrichment analysis applied to high-confidence SMARCA4 targets confirmed neuron related pathways as the top GO Biological processes regulated by SMARCA4 in SCLC. In parallel, SMARCA4 also controls REST, a known suppressor of the NE phenotype, by regulating SRRM4-dependent REST transcript splicing. Furthermore, SMARCA4 inhibition drives ERBB pathway activation in SCLC, rendering SCLC tumors sensitive to afatinib. Conclusions This study nominates SMARCA4 as a key regulator of the NE state plasticity and defines a novel therapeutic strategy for SCLC.
The cell of origin of oncogenic transformation is a determinant of therapeutic sensitivity, but the mechanisms governing cell-of-origin-driven differences in therapeutic response have not been ...delineated. Leukemias initiating in hematopoietic stem cells (HSC) are less sensitive to chemotherapy and highly express the transcription factor
(EVI1) compared with leukemias derived from myeloid progenitors. Here, we compared leukemias initiated in either HSCs or myeloid progenitors to reveal a novel function for EVI1 in modulating p53 protein abundance and activity. HSC-derived leukemias exhibit decreased apoptotic priming, attenuated p53 transcriptional output, and resistance to lysine-specific demethylase 1 (LSD1) inhibitors in addition to classical genotoxic stresses. p53 loss of function in
progenitor-derived leukemias induces resistance to LSD1 inhibition, and EVI1
leukemias are sensitized to LSD1 inhibition by venetoclax. Our findings demonstrate a role for
in p53 wild-type cancers in reducing p53 function and provide a strategy to circumvent drug resistance in chemoresistant
acute myeloid leukemia. SIGNIFICANCE: We demonstrate that the cell of origin of leukemia initiation influences p53 activity and dictates therapeutic sensitivity to pharmacologic LSD1 inhibitors via the transcription factor EVI1. We show that drug resistance could be overcome in HSC-derived leukemias by combining LSD1 inhibition with venetoclax.
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Prior studies have shown that the cell of origin of acute myeloid leukemia (AML) initiation is an important determinant of therapeutic sensitivity. MLL-rearranged leukemias in which the fusion is ...acquired in hematopoietic stem cells (HSC) are less sensitive to chemotherapy and express high levels of the oncogenic transcription factor, Evi1, when compared to leukemias which are initiated in granulocyte-macrophage progenitors (GMP). However, the mechanisms governing how cell-of-origin modulates therapeutic response have not been delineated. Here, we describe a functional link that ties therapeutic sensitivity of MLL-AF9 leukemias to both chemotherapy and pharmacologic inhibitors of lysine-specific demethylase 1 (LSD1) - currently under investigation in clinical trials - to AML cell of origin via a novel mechanism that modulates p53 protein stability and activity through Evi1. This Evi1-dependent mechanism revealed a therapeutic vulnerability in the resistant HSC-derived leukemias that could be targeted with the BCL2 inhibitor venetoclax, which overcame resistance when combined with LSD1 inhibition.
Murine HSC-derived MLL-AF9 leukemias exhibited markedly reduced sensitivity to the LSD1 inhibitor, IMG-7289, when compared to GMP-derived MLL-AF9 leukemias, in vitro and in vivo. Consistent with previously published reports, HSC-derived leukemias exhibited several hundred-fold higher expression of EVI1 mRNA relative to GMP-derived leukemias in both mouse and human MLL-rearranged leukemia models; the differential EVI1 expression mirrors expression patterns of this key hematopoietic regulator in normal HSCs and GMPs. H3K27me3 chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) revealed that the EVI1 locus is silenced by Polycomb repression in GMP-derived leukemias, and this repression was also observed in EVI1low primary AML patient samples. Knockdown of Evi1 via shRNA sensitized HSC-derived leukemias to LSD1 inhibition.
In vitro assays revealed induction of apoptosis in GMP-derived leukemias - but not in HSC-derived leukemias - after treatment with IMG-7289. HSC-derived leukemias also exhibited decreased apoptotic priming assessed through functional BH3 profiling assays as well as blunted p53 transcriptional output. These data suggested that cell-of-origin led to differential p53 activity and therapeutic response in AMLs driven by the same fusion gene initiated from different stem/progenitor populations. The diminished p53 activity in HSC-derived leukemias was associated with reduced p53 protein abundance both at steady-state and after LSD1 inhibitor treatment compared to GMP-derived leukemias. Quantification of p53 mRNA did not show differential p53 expression, including after LSD1 inhibition, in HSC vs. GMP-derived leukemias implicating post-transcriptional regulatory mechanisms that underlie this cell-of-origin mediated phenotype. We found that modulation of Evi1 expression resulted in altered p53 protein stability: specifically, (1) shRNA-mediated knockdown of Evi1 in HSC-derived leukemias increased p53 protein stability and (2) overexpression of Evi1 blunted doxorubicin-induced p53 protein stability. Moreover, p53 loss-of-function in Evi1low GMP-derived MLL-AF9 leukemias induced resistance to LSD1 inhibition. By contrast, Evi1high HSC-derived leukemias exposed to the BCL2 inhibitor venetoclax in vivo were sensitized to LSD1 inhibition, resulting in enhanced apoptosis and greater reductions in disease burden, observations that we observed in patients with Evi1high AML treated with venetoclax. Our findings describe how the cell of origin of p53 wild-type cancers can differentially modulate p53 function and therapeutic response and provide a mechanistic rationale for therapies aimed to circumvent this resistance mechanism.
Cai:Imago Biosciences, Inc.: Consultancy. Goldberg:Celgene: Consultancy; Daiichi-Sankyo: Consultancy, Research Funding; Pfizer: Research Funding; Arog Pharmaceuticals: Research Funding; ADC Therapeutics: Research Funding; American Society of Clinical Oncology: Research Funding; Abbvie: Consultancy; Abbvie: Research Funding; American Society of Hematology: Research Funding; DAVA Oncology: Honoraria. 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Abstract Neuroendocrine (NE) transformation occurs as a mechanism of resistance to targeted therapy in up to 14% and 30% of EGFR-mutant lung and AR-dependent prostate adenocarcinomas, respectively, ...leading to poor prognosis. Even if we know the tumor population at high risk of transformation (TP53/RB1-mutated), no therapies to prevent NE relapse are currently available. To identify therapeutic vulnerabilities for tumors undergoing NE transformation, we performed an in vitro CRISPR screen in a NE-transformed lung tumor model. This screen identified CDC7, involved in DNA replication and DNA damage response, as a potential therapeutic target in this setting. Proteogenomic analyses revealed CDC7 upregulation in lung and prostate clinical samples undergoing NE transformation, detected already in pre-transformation adenocarcinomas. These results indicated that CDC7 expression is induced already at early steps of transformation. Consistently, TP53/RB1-mutated lung and prostate adenocarcinomas exhibited higher CDC7 expression than their double wild-type counterparts. Combined ChIP-seq and promoter reporter assays indicated that CDC7 expression was directly regulated by TP53 and RB1 inactivation. Importantly, TP53/RB1-inactivation induced sensitivity to the CDC7 inhibitor simurosertib, unraveling a therapeutic vulnerability in tumors at high risk of NE transformation. Thus, we tested the combination of simurosertib with targeted therapy in vivo in different lung and prostate patient-derived models of NE transformation, namely TP53/RB1-knock out adenocarcinomas known to undergo NE transformation on targeted therapy. In these, simurosertib was able to suppress NE transformation and dramatically delay tumor relapse. Trajectory analysis on single-cell transcriptomic data for such models revealed a NE transformation transcriptional program occurring already in the untreated tumors before transformation. Remarkably, simurosertib treatment reverted this transcriptomic state and induced a reversion to the original adenocarcinoma transcriptomic profile. CDC7 inhibition led to increased proteasomal activity and degradation of MYC, a stemness transcription factor involved in NE transformation. Ectopic overexpression of MYCT58A, a proteasome degradation-resistant MYC isoform, rescued the NE phenotype in these transformation models, suggesting that CDC7 inhibition-induced MYC degradation is the mechanism by which NE transformation is prevented. In sum, CDC7 inhibition may suppress, or at least dramatically delay NE transformation in patients with lung and prostate adenocarcinomas at high risk of transformation, by inducing MYC proteasomal degradation. The clinical availability of CDC7 inhibitors, currently in phase II clinical trials after demonstrating tolerability and preliminary efficacy, will allow rapid translation of these results into the clinics. Citation Format: Alvaro Quintanal-Villalonga, Fathema Uddin, Kenta Kawasaki, Esther Redin, Vidushi Durani, Amin Sabet, Wouter Karthaus, Yingqian A. Zhan, Samir Zaidi, Moniquetta Shaffer, Harsha Sridhar, Juan Qiu, Parvathy Manoj, Elisa De Stanchina, Michael C. Haffner, Charles L. Sawyers, Charles M. Rudin. CDC7 inhibition prevents neuroendocrine transformation in the lung and prostate through MYC degradation 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 2004.
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