Lynch syndrome (LS) is an inherited predisposition to developing colon, endometrial, ovarian, and other cancers, affecting about one in 300 people. It is caused by inheriting a pathogenic variant in one copy of a DNA mismatch repair (MMR) gene. Cancers that arise in LS patients are MMR deficient, meaning the cancer cells have lost the remaining, functional MMR allele and thereby lost MMR function. It is known that losing MMR function leads to a drastically increased mutation rate. Additionally, cells in culture that lose MMR function become unresponsive to normally lethal DNA damage caused by alkylating agents. However, the question remains whether this immediate, aberrant DNA damage response contributes to cancer development in LS patients. We hypothesize that loss of this MMR-dependent damage response function provides a competitive advantage to colonic progenitor cells. Importantly, we predict that the strength of this advantage depends on the amount and type of DNA-damaging agents in the environment, such as oxidative stress, that promote expansion of MMR-deficient cells. If this hypothesis is true, it would provide insight into how this selective pressure could be mitigated using chemoprevention, and thereby decrease the incidence of colon cancer in this population. To test this, we are using stem cell-derived human colonic organoids and CRISPR gene editing to examine differences in survival and proliferation between otherwise syngeneic MMR-deficient (dMMR) and MMR-proficient (pMMR) colonic organoids. We observe that in atmospheric oxygen, dMMR colonic organoids exhibit enhanced budding compared to pMMR colonic organoids, consistent with our hypothesis of a competitive advantage of dMMR colonic cells. Addition of the antioxidant vitamin C decreases the difference in budding between pMMR and dMMR organoids suggesting this difference is mediated by oxidative stress. Additionally, mixed dMMR and pMMR colonic organoid cultures in ambient oxygen become increasingly dMMR overtime, suggesting dMMR colonic progenitor cells have a competitive advantage over pMMR colonic progenitor cells. To explore the mechanism behind this finding, we are analyzing differences in gene expression between pMMR and dMMR colonic organoids. Preliminarily, we have observed increased expression of the senescent marker gene CDKN2A (p16) and decreased expression of the colonic stem cell marker LGR5 in pMMR organoids compared to dMMR organoids. These results suggest that the MMR pathway may promote senescence and/or terminal differentiation in colonic organoid cultures grown in ambient oxygen. Taken together, these results are consistent with our hypothesis that loss of MMR function imparts a competitive advantage in colonic cells in non-transformed colonic organoids that could contribute to cancer formation in LS patients. Caroline Guild, Abhijit Rath, Christopher Heinen. Investigating competitive advantage of mismatch repair loss in colonic organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6711.

Abstract Lynch syndrome (LS) is the most common inherited predisposition for developing colon cancer and is caused by germline variants in mismatch repair (MMR) genes. Still, the mechanism of tumorigenesis in these patients is unknown. An LS patient inherits one defective copy of an MMR gene. At some point in their lifetime, the remaining wild-type allele is lost in a cell rendering it entirely MMR deficient. MMR deficiency increases mutational rate and the chance of acquiring mutations in an oncogene or tumor suppressor that drive tumor initiation. However, loss of MMR in cells also leads to increased resistance to certain forms of DNA damage. Thus, our question is whether this phenotype imparts a selective advantage in colonic cells that may lead to clonal expansion of MMR-deficient cells as a first step towards tumorigenesis in LS patients. To test this, we are using stem cell-derived human colonic organoids and CRISPR gene editing to examine competition between MMR-deficient (MMRd) and MMR-proficient (MMRp) colonic cells over time. We have initially observed that MMRd stem cells are much more efficient at generating colonic organoids that grow larger and survive longer, consistent with our hypothesis of a selective advantage. To mimic the spontaneous loss of heterozygosity that occurs in LS patients’ colonic crypts, we are forming organoids from differentially labeled MMRd and MMRp stem cells. Live fluorescent imaging of individual organoids is used to quantify changes in cell population over time. Preliminary data indicate that MMRd cells can outgrow MMRp cells resulting in a shift in the ratio of green to red cells over a two-week time period while challenged with a DNA-damaging agent. These results suggest that loss of MMR function imparts an immediate selective advantage in colonic cells in response to DNA damage. Citation Format: Caroline Guild, Christopher Heinen, Samantha Nadeau. An organoid model for investigating Lynch syndrome tumorigenesis [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 258.

Lynch syndrome (LS) is an inherited predisposition for developing cancer caused by germline variants in mismatch repair (MMR) genes. It is estimated that LS is responsible for 1 out of every 35 colorectal cancers and 1 out of every 56 endometrial cancers. Loss of MMR is also seen in many sporadic cancers. Still, the mechanism of tumorigenesis in these patients is unknown. Loss of MMR increases genomic mutation rate, but it is unclear if increased mutational burden alone leads to tumorigenesis. Recent studies of LS patient colon samples show fields of morphologically normal tissue in which cells have lost expression of an MMR protein, suggesting that loss of MMR in these cells is sufficient to confer an advantage over heterozygous cells still proficient in MMR. However, whether MMR loss provides a direct advantage to cell types associated with cancer in LS has not been directly tested. We hypothesize that loss of MMR is sufficient to confer a selective advantage to colonic stem cells and is the first step towards tumorigenesis in LS patients. To test this, we use a human colonic organoid model to examine competition between MMR-deficient (dMMR) and MMR-proficient (pMMR) cells over time. The colonic organoids are differentiated from human embryonic stem cells. To track cell populations, dMMR stem cells are labeled with mCherry and pMMR stem cells are labeled with GFP. We have initially observed that dMMR stem cells are much more efficient at generating colonic organoids. Thus, the cells are mixed so that resulting organoids initially contain a small population of dMMR cells in order to mimic the spontaneous loss of heterozygosity that occurs in LS patients’ colonic crypts. Live fluorescent imaging of individual organoids is used to quantify changes in cell population over time. Preliminary data indicate that dMMR cells can outgrow pMMR cells resulting in a shift in the ratio of green to red cells over a two-week time period. These results suggest that loss of MMR function imparts an immediate selective advantage. Furthermore, this assay could be used to find conditions or compounds that decelerate population shifts with the ultimate goal of finding a way to reduce dMMR fields in LS patients. In these ways, this assay can be leveraged for gaining new insights into MMR function, pathogenesis of LS, and potential therapies for LS. Citation Format: Caroline Guild, Kirby Madden-Hennessey, Chris Heinen. An organoid model for understanding the pathogenesis of Lynch syndrome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 149.

Pancreatic ductal adenocarcinomas (PDACs) frequently harbor KRAS mutations. Although MEK inhibitors represent a plausible therapeutic option, most PDACs are innately resistant to these agents. Here, we identify a critical adaptive response that mediates resistance. Specifically, we show that MEK inhibitors upregulate the anti-apoptotic protein Mcl-1 by triggering an association with its deubiquitinase, USP9X, resulting in acute Mcl-1 stabilization and protection from apoptosis. Notably, these findings contrast the canonical positive regulation of Mcl-1 by RAS/ERK. We further show that Mcl-1 inhibitors and cyclin-dependent kinase (CDK) inhibitors, which suppress Mcl-1 transcription, prevent this protective response and induce tumor regression when combined with MEK inhibitors. Finally, we identify USP9X as an additional potential therapeutic target. Together, these studies (1) demonstrate that USP9X regulates a critical mechanism of resistance in PDAC, (2) reveal an unexpected mechanism of Mcl-1 regulation in response to RAS pathway suppression, and (3) provide multiple distinct promising therapeutic strategies for this deadly malignancy.

Supplementary Figures and Legends

Lynch syndrome (LS) is the most common hereditary form of colon cancer, resulting from a germline mutation in a DNA mismatch repair (MMR) gene. Loss of MMR in cells establishes a mutator phenotype, which may underlie its link to cancer. Acquired downstream mutations that provide the cell a selective advantage would contribute to tumorigenesis. It is unclear, however, whether loss of MMR has other consequences that would directly result in a selective advantage. We found that knockout of the MMR gene MSH2 results in an immediate survival advantage in human stem cells grown under standard cell culture conditions. This advantage results, in part, from an MMR-dependent response to oxidative stress. We also found that loss of MMR gives rise to enhanced formation and growth of human colonic organoids. These results suggest that loss of MMR may affect cells in ways beyond just increasing mutation frequency that could influence tumorigenesis.

While KRAS mutations are common in non-small cell lung cancer (NSCLC), effective treatments are lacking. Here, we report that half of KRAS-mutant NSCLCs aberrantly express the homeobox protein HOXC10, largely due to unappreciated defects in PRC2, which confers sensitivity to combined BET/MEK inhibitors in xenograft and PDX models. Efficacy of the combination is dependent on suppression of HOXC10 by BET inhibitors. We further show that HOXC10 regulates the expression of pre-replication complex (pre-RC) proteins in sensitive tumors. Accordingly, BET/MEK inhibitors suppress pre-RC proteins in cycling cells, triggering stalled replication, DNA damage, and death. These studies reveal a promising therapeutic strategy for KRAS-mutant NSCLCs, identify a predictive biomarker of response, and define a subset of NSCLCs with a targetable epigenetic vulnerability.

While the majority of BRAF-mutant melanomas respond to BRAF/MEK inhibitors, these agents are not typically curative. Moreover, they are largely ineffective in NRAS and NF1-mutant tumors. Here we report that genetic and chemical suppression of HDAC3 potently cooperates with MAPK pathway inhibitors in all three Ras pathway-driven tumors. Specifically, we show that entinostat dramatically enhances tumor regression when combined with BRAF/MEK inhibitors, both in models that are sensitive or relatively resistant to these agents. Interestingly, MGMT expression predicts responsiveness and marks tumors with latent defects in DNA repair. BRAF/MEK inhibitors enhance these defects by suppressing homologous recombination genes, inducing a BRCA-like state; however, entinostat addition triggers the concomitant suppression of NHEJ genes, resulting in a chemical synthetic lethality caused by excessive DNA damage. Together these studies identify melanomas with latent DNA repair defects, describe a promising drug combination that capitalizes on these defects, and reveal a tractable therapeutic biomarker. Citation Format: Ophélia Maertens, Ryan Kuzmickas, Haley Manchester, Chloe Emerson, Alessandra Gavin, Caroline Guild, Terence Wong, Thomas De Raedt, Christian Bowman-Colin, Elodie Hatchi, Levi Garraway, Keith Flaherty, Shailja Pathania, Stephen Elledge, Karen Cichowski. MAPK pathway suppression unmasks latent DNA repair defects and confers a chemical synthetic vulnerability inBRAF,NRASandNF1-mutant melanomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-113.

Abstract Although the majority of BRAF-mutant melanomas respond to BRAF/MEK inhibitors, these agents are not typically curative. Moreover, they are largely ineffective in NRAS- and NF1-mutant tumors. Here we report that genetic and chemical suppression of HDAC3 potently cooperates with MAPK pathway inhibitors in all three RAS pathway–driven tumors. Specifically, we show that entinostat dramatically enhances tumor regression when combined with BRAF/MEK inhibitors, in both models that are sensitive or relatively resistant to these agents. Interestingly, MGMT expression predicts responsiveness and marks tumors with latent defects in DNA repair. BRAF/MEK inhibitors enhance these defects by suppressing homologous recombination genes, inducing a BRCA-like state; however, addition of entinostat triggers the concomitant suppression of nonhomologous end-joining genes, resulting in a chemical synthetic lethality caused by excessive DNA damage. Together, these studies identify melanomas with latent DNA repair defects, describe a promising drug combination that capitalizes on these defects, and reveal a tractable therapeutic biomarker. Significance: BRAF/MEK inhibitors are not typically curative in BRAF-mutant melanomas and are ineffective in NRAS- and NF1-mutant tumors. We show that HDAC inhibitors dramatically enhance the efficacy of BRAF/MEK inhibitors in sensitive and insensitive RAS pathway–driven melanomas by coordinately suppressing two DNA repair pathways, and identify a clinical biomarker that predicts responsiveness. See related commentary by Lombard et al., p. 469. This article is highlighted in the In This Issue feature, p. 453