Mutant Mice Research Articles

Effect of inactivating heterozygous mutations in DNA repair genes on experimental lung carcinogenesis in mice

Background. Inactivating mutations in Chek2 and Gprc5a genes are known to be associated with cancer development. Experimental carcinogenesis studies in genetically modified mice generate new data on their influence on pathology development.Aim. In the present study in a model of lung carcinogenesis, survival parameters as well as tumor multiplicity and size in mice with Chek2 and Gprc5a heterozygous inactivating mutations were evaluated.Material and methods. F2 hybrid mice from crosses between CBAB6F1 males heterozygous for the studied mutations and wild-type BALB / c females were used: Chek2dAA-carriers (76 males and 64 females) and Gprc5ainsA-carriers (60 males and 42 females). Starting at four months of age, mice received urethane (ethyl carbamate) intraperitoneally at a dose of 600 mg / kg weekly for 6 weeks. After genotyping by allele-specific PCR, animals were allocated to groups. Carcinogenesis parameters were evaluated 40 weeks after the beginning of the experiment.Results. The proportion of mice with mutations surviving to the age of three months roughly followed the Mendelian distribution (35 / 41 males and 33 / 31 females) for the offspring of males heterozygous for Chek2dAA and was significantly lower in the case of Gprc5ainsA (20 / 40 males and 17 / 25 females, p = 0.043). The death of Gprc5ainsA carriers during the experiment was also higher than in the control group (p = 0.0506 in females). Synchronous lung and thymus neoplasms were found in 2 out of 4 Gprc5ainsA females that died before the end of the experiment, which were not found in other groups. At the end of the experiment, no significant differences in tumor multiplicity, mean linear size, and volume were found between the groups of mice with and without mutations.Conclusion. It was found that heterozygous inactivating mutation Chek2dAA does not affect early age development and does not modify the parameters of induced lung carcinogenesis in mice. Heterozygous carriage of Gprc5ainsA mutation in mice increases the risk of early death and sensitivity to the toxic and carcinogenic effects of urethane.

Abstract 5538: Efferocytosis-induced HIF-1α enhances tumor vascularization and promotes osteoblastic bone remodeling in metastatic prostate cancer

Abstract Metastatic prostate cancer (PCA) exhibits a predilection for bone metastasis, initiating a complex interplay with bone cells, leading to dysregulated remodeling and mixed-pathological bone alterations. Human PCA primarily yields osteoblastic bone lesions, linked to bone pain and an adverse prognosis. In this study, we explored the activation of HIF-1α in myeloid phagocytic cells by apoptotic cancer cells in the context of bone metastatic PCA. Our previous findings demonstrated HIF-1α stabilization in bone macrophages engulfing apoptotic PCA cells in vitro. This study extends our understanding to an in vivo setting using mouse prostate cancer cells designed to express inducible caspase 9, triggering apoptosis (and subsequent efferocytosis) when exposed to the dimerizer molecule AP20187 (AP). Our assessment of PCA tumor growth in bone utilized a syngeneic mouse model, introducing a loss-of-function Hif1a mutation into myeloid lineage cells using LysM-Cre mice crossed with Hif1a-floxed mice (Hif1amut (LysM-Cre+/- Hif1aflox)), compared with the LysM-Cre-/- Hif1aflox (WT). In the first model, cancer cells were inoculated into the tibiae of these mice and apoptosis was induced with AP on days 3 and 5 post-inoculation (AP model). Results were compared to a model where apoptosis was not induced after inoculation (No-AP). In the AP model, fractional bone volume (BV/TV) in the medullae was significantly higher in tumor-inoculated tibiae than in non-inoculated (No-tumor) contralateral bone, highlighting the predominance of osteoblastic lesions. Conversely, in the No-AP model, increased BV/TV significance was achieved in WT mice (tumor vs. No-tumor), but not in mutant mice. Trabecular thickness (Tb.Th) was increased in both models in the presence of tumor, regardless of the Hif1a phenotype. Importantly, significant differences in BV/TV and Tb.Th between WT and Hif1amut tumor tibiae were exclusively noted in the context of induced apoptosis. Histological differences between tumors in the AP model were observed by immunofluorescence (IF) staining, which revealed a reduced fractional F4/80+ area in Hif1amut vs. WT tumors, with no differences in TRAP+ (osteoclasts) lining bone surfaces. RNAseq analysis of HIF1amut vs. WT macrophages efferocytosing apoptotic cancer cells showed downregulated processes related to vasculature development and blood vessel morphogenesis (p< 10-5). In correlation, IF analysis with endomucin (EMCN) demonstrated a reduction in stained area (EMCN+) relative to tumor area in Hif1amut tibiae vs. WT, suggesting a role of efferocytosis-induced HIF-1α in tumor vascularization. This study unveils intricacies of metastatic tumor growth in bone and proposes a connection between efferocytosis-induced HIF-1α in myeloid phagocytic cells and the promotion of tumor vascularization, alongside the enhancement of osteoblastic bone remodeling. Citation Format: Gabriel G. Kleer, Maria Molina Sanchez, Veronica Mendoza-Reinoso, Amy J. Koh, John Rubin, Laurie K. McCauley, Hernan Roca. Efferocytosis-induced HIF-1α enhances tumor vascularization and promotes osteoblastic bone remodeling in metastatic prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5538.

Abstract 6272: Loss of SIRT6 diverts TGF-β signaling to promote liver cancer

Abstract Background: In the US, ~35% of individuals on the waitlist for a liver transplant who had hepatocellular cancer (HCC) also had metabolic dysfunction-associated steatohepatitis (MASH). Yet, key signaling mechanisms controlling cellular transformation in this obesity-driven HCC remain only partially understood. SIRT6 is NAD+-dependent protein deacetylases that limits the activity of the lipogenic transcription factors SREBP1/2. Reduced SIRT6 is associated with human fatty liver and cancer. However, SIRT6 also limits liver fibrosis by inhibiting TGF-β signaling through the deacetylation of SMAD2 and SMAD3. We found that Smad3 driven TGF-β signaling is modulated by βII-Spectrin (encoded by SPTBN1), and that a hot mutation in HCC- D1089Y encompasses the SPTBN1-SMAD3 binding site (Gastro 2018; 154:195-210). In stressed hepatocytes, βII-Spectrin promotes lipogenesis through interactions between caspase-cleaved forms of both SREBP1 and βII-Spectrin (Sci Transl Med 2021;13:624). Liver-specific knockout of βII-Spectrin (LSKO) blocks fatty liver and HCC. Here we explored the extent of βII-spectrin-D1089Y regulation of SIRT6 expression through SMAD3, and the combined roles of SIRT6 and SPTBN1 in human HCC. Methods: We generated a SPTBN1-D1089Y mutant mouse and examined SIRT6 in the SPTBN1-D1089Y mutant mouse liver tissues and MEFs. TGF-β/Smad3 pathway activity was analyzed in SPTBN1-D1089Y mutant MEFs via western blot and RNA-seq. SIRT6 expression was examined in mouse MAFLD/MASH (GSE156059). Structure-based molecular docking simulations using three-dimensional structural coordinates of human SIRT6 (ID: Q8N6T7) and β2SP (ID: Q01082) from the AlphaFold database, followed by interactions between SIRT6 and SPTBN1. Results: SIRT6 levels are decreased in SPTBN1-D1089Y mutant liver cancer cell line and mouse liver tissues. TGF-β/SMAD3 pathway activity was altered in SPTBN1-D1089Y mutant MEF cells with decreased phospho-SMAD3 levels. The structural model predicted specific interactions between human SPTBN1 and SIRT6. Overexpression of Flag-SIRT6 reduced βII-Spectrin acetylation in HepG2 cells. TGF-β induced SMAD3, βII-Spectrin coimmunoprecipitation with SIRT6. SIRT6 knockdown increases nSREBP1, SCD1 (encoded by a nSREBP1 target gene), and βII-Spectrin in HepG2 cells. SIRT6 RNA expression is significantly decreased in liver macrophages from mice with NAFLD/NASH. Conclusions: Our data indicated a SMAD dependent as well as a SMAD3 independent role of SPTBN1 in regulating SIRT6. βII-spectrin-D1089Y together with SIRT6 deficiency may increase the susceptibility to MASH/HCC via regulating the crosstalk between macrophage and hepatocytes or regulating MASH and HCC. Citation Format: Kazufumi Ohshiro, Xiyan Xiang, Xiaochun Yang, Krishanu Bhowmick, Mohammad I. Hassan, Taj Mohammad, Lopa Mishra. Loss of SIRT6 diverts TGF-β signaling to promote liver cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6272.

Abstract 771: A TGF-β superfamily member, myostatin contributes to MASH and HCC via muscle atrophy by disrupting TGF-β signaling, and is a potential marker with PKM2 for human HCC

Abstract Background: In patients with cirrhosis and hepatocellular carcinoma (HCC), sarcopenia or skeletal muscle loss, as well as metabolic alterations are frequent and progressive comorbidities. Myostatin, a TGF-β superfamily member is expressed primarily in skeletal muscle, inhibits muscle growth, and causes sarcopenia in cirrhosis. Previously, we had observed liver-specific knockout of the SMAD3 adaptor, βII-spectrin -Sptbn1 (βS-LSKO) or siRNA targeting βII-spectrin blocks sarcopenia, fatty liver disease-MASH and HCC (Sci TM 2021), suggesting that βII-spectrin may be a viable strategy to identify mechanisms, as well as new biomarkers of MASH and HCC. Methods: 1. SPTBN1Flox (control) and liver-specific βII-spectrin knockout (SPTBN1LSKO) mice were fed Western diet (WD) and treated with Diethylnitrosamine (DEN) for obesity-associated HCC. Muscle mass, body weight and multiple enzymes involved in glycolysis as well as sarcopenia were measured in mutant mice. 2. In tumors, a rate-limiting enzyme in glycolysis, pyruvate kinase isozyme M2 (PKM2) that mediates inflammation and aerobic glycolysis in Warburg effect. Therefore, we further examined TGF-β pathway-enriched biomarkers such as Myostatin and PKM2 for markers of HCC and whether these could stratify risk in patients with cirrhosis for HCC, in a four-institution cohort, (n=157) compared to cirrhosis patients (n=380). Results: 1. Like human fatty liver disease, obese mutant mice show marked sarcopenia with increased PKM2 and Myostatin levels with decreased p-RPS6/total-RPS6 ratio (indicating mTOR signaling inhibition) in striated muscle tissue. In contrast, levels are restored to normal in the liver specific knockout of SPTBN1. 2. Using the 108 markers as well as PKM2 and myostatin (MSTN), we found 31 markers with false discovery rate (FDR) p values < 0.10 from univariable Kruskal-Wallis tests for the associations of markers with HCC vs. cirrhosis. Conclusions: Our results reveal that SMAD3 adaptor, βII-spectrin liver-specific knockout decreases Myostatin and PKM2 expression, providing new insight into molecular mechanisms in MASH and HCC. These studies reflect that these are biologically functional circuits that could provide new serum markers, such as Myostatin and PKM2 for risk stratification of HCC. Citation Format: Kazufumi Ohshiro, Krishanu Bhowmick, Xiyan Xiang, Shivani Kapoor, Richard Amdur, Srinivasan Dasarathy, Adrian R. Krainer, Lopa Mishra. A TGF-β superfamily member, myostatin contributes to MASH and HCC via muscle atrophy by disrupting TGF-β signaling, and is a potential marker with PKM2 for human HCC [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 771.

Abstract 1442: Establishing a papillary renal cell carcinoma mouse model with activating Met mutation

Abstract Renal cell carcinoma (RCC) is a heterogeneous group of cancers of the nephron. Papillary renal cell carcinoma (PRCC) is the second most common type of RCC. c-Met activating mutations have been associated with Type 1 PRCC which are made up of small cells containing basophilic cytoplasm covering papillae and tubular structures. In patients with PRCC, c-MetH1112Y missense mutations are associated with both somatic and germline mutations. Previous functional studies indicate codon H1112 plays an important structural role in maintaining c-Met in the inactive conformation and alterations at this codon, including H1112Y, likely disrupt this interaction facilitating constitutive activation with increased c-MET activity. A c-MetH1112Y (c-MetHY) transgene with tetO promoter sequence was micro-injected into the mouse oocyte. Transgenic mice carrying this transgene have the ability to express this activated Met protein in cells expressing rtTA (reverse tetracycline-controlled transactivator) and when doxycycline is present. Mutant mice harboring this transgene along with additional trp53 mutations showed glomeruloid like Type 1 PRCC. Our in vivo studies confirmed the expression of c-MetHY gene within the kidney and Lrp2 and Ki67 staining indicated that PRCC arises from PT (proximal tubule). PRCC canonical markers CK7, AMACR and MET expression confirmed the Type1 PRCC in our mouse model. Additionally Pan-CK, E-Cad positive expression in mutant mice further confirmed PRCC arises from epithelial cells. We have generated and described type 1 PRCC mouse model with activating-MetHY mutation. This mouse model will help illustrate the oncogenic mechanism of Met activation and identify the critical events during progression of PRCC. Citation Format: Preet Lal, Li Ding, Feng Chen. Establishing a papillary renal cell carcinoma mouse model with activating Met mutation [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 1442.

Abstract 1970: Discovery and characterization of potent and selective HER2 exon20 insertion mutant inhibitors

Abstract Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, accounting for over 85% of all cases. Among NSCLC, approximately 2-4% of patients have mutations in the HER2, a member of the ErbB family of receptor tyrosine kinases and key proliferation driver, with exon 20 insertions making up about 90% of these mutations. HER2 exon mutations can have various alterations, and among them, the most common HER2 mutation is in-frame insertions in exon 20, especially the A775_G776insYVMA insertion/duplication. These mutations can affect the structure and function of the HER2 protein and may contribute to tumor development and growth. HER2 mutations have often shown resistance to EGFR tyrosine kinase inhibitors (TKIs), further highlighting the need for HER2-specific treatments. Also, inhibitors targeting HER2 mutations are selective, meaning they specifically target HER2 alterations without causing toxicities or affecting EGFRWT. Therefore, there is a need for the development of selective HER2 exon20 insertion mutant inhibitors. Herein, Hanmi has identified selective HER2 exon20 insertion mutant small molecule inhibitors, designed based on affinity and binding mode starting from a known crystal structure using GLIDE (Schrödinger), and the screening for the viability of cell lines including HER2A775_G776insYVMA, HER2G776delinsVC , HER2WT and EGFRWT was investigated. Hanmi's HER2 exon20 insertion mutant inhibitors selectively target mutant HER2 receptor tyrosine domains through shared binding, sparing EGFRWT and minimizing EGFR-related toxicities. With highly selectivity against EGFRWT and desirable pharmacokinetic properties, the Hanmi compounds inhibit HER2 mutant cells and HER2 mutant enzymes. Additionally, the compounds have an oral administration-compatible DMPK profile and demonstrated anti-tumor activity in HER2 mutant and HER2WT tumor xenograft mice. In conclusion, based on our study, Hanmi compounds, a novel HER2 exon20 insertion mutant inhibitors could be suggested as an appropriate therapeutic agent for inhibitory activity against HER2 exon20 insertion mutant NSCLC. Citation Format: Ho Yeon Nam, Sun Young Jang, Jiyoung Jeon, HyungSeok Yoo, Jooyun Byun, Gunwoo Lee, Youngjoo Lee, Yu-Yon Kim, Soye Jeon, Young Gil Ahn. Discovery and characterization of potent and selective HER2 exon20 insertion mutant inhibitors [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 1970.

Abstract 6610: Metabolic patterns of pancreatic cancer cachexia: Cross-tissue lipid networks predict cachexia progression

Abstract Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal of all cancer types. A key — yet poorly understood — facet in the disease state is cachexia, a multi-organ pathological state characterized by physical wasting and tissue catabolism. It occurs in 80% of PDAC patients, and to date there are no preventative or early detection methods. Cachexia leads to limited tolerance to anti-cancer therapy and contributes to disease lethality. Here, we present the first-of-its-kind systemic metabolomic analysis across cachectic stages to better understand disease progression. We used the well-established mutant KRASG12D(LSL/+) mutant p53 inducible mouse model of PDAC. Model physiology faithfully recaptures human cachexia: we observe progressive overall weight loss as well as loss of skeletal muscle and adipose tissue. As with human disease, weight loss occurs prior to loss of appetite in the animals, suggesting physiological changes independent of nutrition. Importantly, our model captures the period prior to weight loss (pre-CAC), as well as early-CAC (<10% weight loss) and late-CAC (>10% weight loss). We profiled the metabolomes of the pancreas, interstitial fluid, plasma, liver, 3 different adipose tissues, and 3 skeletal muscles, from male and female mice, control and tumor-bearing, across all 3 stages of cachexia. Each tissue has a unique metabolic trajectory across cachexia stages; pathway and correlation analyses showed a particular emphasis on lipid alterations in peripheral tissues. Strikingly, we find systemic metabolic changes prior to tissue wasting, including rewiring in the liver before the presence of metastases. This indicates systemic alterations as early as pre-CAC. We therefore used computational modeling to identify metabolites that may be participating in cross-tissue networks. A key finding, confirmed by 13C tracing, was the circulation of lactate and glucose and their uptake by the liver and pancreas. Network maps also suggest lipid rewiring in distal tissues, changes in abundances of these same lipid species in plasma, and their consequent alterations in the tumor and liver. A fundamental question remains: can we predict cachexia development before the manifestation of symptoms? We used feature selection algorithms based on statistical learning, training the models only on a subset of data. Specific sphingolipids and triglycerides were the key lipid species that distinguished control and pre-CAC. We find that the model — trained only on pre-CAC — is able to determine early-CAC with an 85% accuracy and late-CAC with a 90% accuracy. Thus, a limited set of metabolites that are altered in pre-CAC could have predictive value for future cachexia development. Overall, our work provides a resource for the field and advances our understanding of systemic metabolism in PDAC cachexia. We hope this will lay the foundation for cachexia prevention and treatment. Citation Format: Deepti Mathur, Blanca Majem, Courtney Beaulieu, Lucas Dailey, Sarah Jeanfavre, Joao Xavier, Clary Clish, Nada Kalaany. Metabolic patterns of pancreatic cancer cachexia: Cross-tissue lipid networks predict cachexia progression [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 6610.

Abstract 2667: Mucin 5AC inhibition via intratracheal RNAi delivery as an effective alternative strategy for prevention and treatment of Kras mutant lung cancer

Abstract Lung cancer, including lung adenocarcinoma (LUAD), is the leading cause of cancer-related deaths in the United States. KRAS mutations are the most prevalent oncogenic driver alteration in human LUAD. We have previously shown that high expression of Mucin 5 AC (MUC5AC), a main airway secretory mucin, is significantly associated with poor prognosis. Mucins are essential for mucociliary clearance and the maintenance of airway homeostasis, however, their overproduction could negatively affect the lung immune microenvironment and has been shown to promote airway diseases such as chronic obstructive pulmonary disease (COPD) and lung cancer. Previously, we have shown that genetic deletion of MUC5AC in a murine model of K-ras mutant lung adenocarcinoma (KM-LUAD) impedes tumor development and reduces pro-tumor inflammatory and immune phenotypes. These findings indicate that MUC5AC is a potential druggable target against KM-LUAD. In this study, we sought a translational approach and inhibited MUC5AC in our K-rasG12D mutant mouse model of LUAD via RNA interference (RNAi). Six-week-old mice were given RNAi (5mg/kg) or vehicle (saline) via biweekly intratracheal administration for 8 weeks. This approach led to a significant decrease in tumor burden (40%) and a trending increase in total white blood cell counts, predominantly macrophages in the lung tumor microenvironment which was associated with phenotypic changes in macrophages characterized by reduced expression of Fizz1, and M2 pro-tumor macrophage marker. Additionally, RNAi-treated mice had reduced expression of Siglec-E, a mucin receptor and known inhibitor of the TLR4 endocytosis and promoter of NF-ĸB signaling, as well as a significant decrease in IL-6, a product of the NF-ĸB pathway and promoter of STAT3 signaling which we have previously shown to have an essential role in promotion of KM-LUAD. In summary, these findings elucidate a potential mechanism by which MUC5AC protects tumor cells from apoptosis and immune-mediated cytotoxic activity but also highlights how MUC5AC RNAi is an effective indirect preventive and therapeutic strategy in high-risk individuals (smokers with COPD) and patients with early stage KM-LUAD, respectively. Citation Format: Melvin Zarghooni, Maria T. Grimaldo, Avantika Krishna, Michael J. Clowers, Linda Phan, Yasmina H. Rezai, Arnav Gaitonde, Carlos Rodriguez Reyna, Walter Velasco Torrez, Erik Bush, Seyed Javad Moghaddam. Mucin 5AC inhibition via intratracheal RNAi delivery as an effective alternative strategy for prevention and treatment of Kras mutant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2667.

Abstract 5949: The clinical dual KRASG12C inhibitor FMC-376 has demonstrated potential as both a monotherapy and in combination for the treatment of patients with KRASG12C mutation positive NSCLC

Abstract KRAS, a membrane-associated guanosine triphosphatase (GTPase), is the most frequently mutated oncogene in cancer with activating mutations occurring in approximately 25% of NSCLC. Of these, KRASG12C occurs in approximately 14% of adenocarcinomas and in 0.5 to 4% of squamous NSCLCs. This mutation impairs GTPase activity and GTP-hydrolysis leading to an increase in the active, GTP-bound (ON), state. KRAS was long considered undruggable until the recent discovery of inhibitors that bind the GDP-bound, inactive (OFF) state of KRASG12C. The most clinically advanced of these first-generation molecules have demonstrated clinical response rates of 37-43% and 6 to 7-month progression-free survival in patients with KRASG12C positive NSCLC. While significant, a majority of cancers do not respond, and acquired resistance is common. FMC-376 is a next-generation dual inhibitor of both the ON and OFF states of KRASG12Cwith the potential to deliver improved outcomes as both a monotherapy and in combination, for patients with KRASG12C positive NSCLC. FMC-376 was discovered through the FrontierTM platform, which integrates chemoproteomics, machine-learning, and covalent fragment-based drug discovery. The potential for FMC-376 in the treatment of patients with KRASG12C mutant NSCLC has been assessed in a broad panel of NSCLC PDX models representing settings of sotorasib/adagrasib resistance, in a model of NSCLC CNS-metastasis and in combination with an immune checkpoint inhibitor. The dual ON + OFF state mechanism of action of FMC-376 is highly active across a panel of NSCLC CDX and PDX models, including those that carry co-mutations that are associated with both primary and acquired resistance to the OFF-state inhibitors adagrasib and sotorasib in NSCLC. These drivers of resistance include activating mutations of receptor tyrosine kinases (e.g. EGFR, MET, etc.), co-mutations including KEAP1 and adaptive resistance due to release of negative feedback control. Evaluation of FMC-376 in models where EGFR signaling is induced by EGF demonstrated rapid and durable target engagement in contrast to both sotorasib and adagrasib which show decreased effectiveness after EGF stimulation. As a result, FMC-376 is highly effective in NSCLC PDX models that have EGFR and MET upregulation. In addition, 27-42% of patients with KRASG12C positive NSCLC present with CNS metastasis. FMC-376 is highly active in an intracranial NSCLC model of CNS-metastasis, resulting in tumor regression. Furthermore, FMC-376 has demonstrated activity in combination with an anti-PD-1 mAb in a KRASG12C mutant mouse syngeneic tumor model. Together, these data demonstrate the potential of FMC-376, a next-generation clinical stage dual inhibitor of ON and OFF state KRASG12C, for the treatment of patients with KRASG12C mutant NSCLC as well as other KRASG12C positive tumors. Citation Format: Yan Wang, Allison Roberts, Philamer Calses, Richard M. Neve, Jocelyn Staunton, Kevin R. Webster. The clinical dual KRASG12C inhibitor FMC-376 has demonstrated potential as both a monotherapy and in combination for the treatment of patients with KRASG12C mutation positive NSCLC [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 5949.

Transcription elongation defects link oncogenic SF3B1 mutations to targetable alterations in chromatin landscape

Transcription and splicing of pre-messenger RNA are closely coordinated, but how this functional coupling is disrupted in human diseases remains unexplored. Using isogenic cell lines, patient samples, and a mutant mouse model, we investigated how cancer-associated mutations in SF3B1 alter transcription. We found that these mutations reduce the elongation rate of RNA polymerase II (RNAPII) along gene bodies and its density at promoters. The elongation defect results from disrupted pre-spliceosome assembly due to impaired protein-protein interactions of mutant SF3B1. The decreased promoter-proximal RNAPII density reduces both chromatin accessibility and H3K4me3 marks at promoters. Through an unbiased screen, we identified epigenetic factors in the Sin3/HDAC/H3K4me pathway, which, when modulated, reverse both transcription and chromatin changes. Our findings reveal how splicing factor mutant states behave functionally as epigenetic disorders through impaired transcription-related changes to the chromatin landscape. We also present a rationale for targeting the Sin3/HDAC complex as a therapeutic strategy.

Abstract 593: DCC-3084, a brain penetrant RAF dimer inhibitor, broadly inhibits BRAF class I, II, and III alterations leading to growth inhibition of intracranially implanted tumors in preclinical models

Abstract Background: Mutations in the RAS/MAPK pathway are a frequent driver of cancer, with oncogenic RAS or RAF mutations occurring in >30% of all cancers. First generation BRAF inhibitors are approved for use for tumors with Class I BRAF mutations (V600X). However, these drugs are not efficacious in RAF dimer mutant and RAS mutant cancers due to paradoxical activation of RAF dimers. Herein, we describe DCC-3084, a brain penetrant, potent and selective investigational switch-control inhibitor of BRAF and CRAF kinases, shown preclinically to target all relevant aberrant signaling mechanisms (monomers, heterodimers and homodimers) and inhibits growth of subcutaneously and intracranially implanted tumors in preclinical models. Methods: Inhibition of RAF kinases was measured using recombinant enzymes. Cellular proliferation was measured using resazurin to monitor cell viability. Inhibition of ERK or RSK phosphorylation was measured by AlphaLISA or ELISA. Pharmacokinetics (PK) in the plasma, brain and CSF compartments were measured following oral dosing in Wistar rats. Subcutaneous or intracranial BRAF mutant mouse xenograft models were used to assess PK, pharmacodynamics (PD), and efficacy. Results: DCC-3084 treatment resulted in potent single-agent inhibition of MAPK pathway signaling, and cellular proliferation in a wide range of Class I, II, III BRAF and BRAF fusion altered cell lines. DCC-3084 was demonstrated to be CNS penetrable and exhibited dose dependent oral exposure with robust inhibition of the MAPK pathway in PK/PD models. Oral treatment of DCC-3084 as a single agent resulted in tumor regression in subcutaneously implanted BRAF Class I and BRAF fusion xenograft models. Additionally, DCC-3084 resulted in tumor regression or growth inhibition in intracranially implanted BRAF Class I and BRAF fusion tumor models. Conclusions: The switch-control inhibitor DCC-3084 broadly inhibits Class I, II and III BRAF mutations, BRAF fusions, and BRAF/CRAF heterodimers leading to tumor regression in preclinical models. DCC-3084 CNS penetration enables potential for further research in brain metastases or primary brain cancer. Citation Format: Gada Al-Ani, Stacie L. Bulfer, Jefferey D. Zwicker, Chase K. Crawley, Kristin M. Elliot, Salim Javed, Qi Groer, Molly M. Hood, Joshua W. Large, Kylie Luther, Yeni K. Romero, Forrest A. Stanley, Daniel C. Tanner, Hanumaiah Telikepalli, Bertrand Le Bourdonnec, Bryan D. Smith, Daniel L. Flynn. DCC-3084, a brain penetrant RAF dimer inhibitor, broadly inhibits BRAF class I, II, and III alterations leading to growth inhibition of intracranially implanted tumors in preclinical models [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 593.

Abstract 5263: The impact of MRTX1133 on anti-tumor immunity in lung cancer with KRASG12D mutation

Abstract Background: To date, there have been no clinical molecules that are selective for KRASG12D mutant tumors. Although immune checkpoint inhibitors (ICIs) have improved the survival for patients with lung cancer, inferior efficacy of ICIs has been reported in KRASG12D mutant NSCLC. MRTX1133 is a selective KRASG12D inhibitor, which has been investigated in early clinical studies. In this study, we aimed to reveal the immune status using preclinical murine model of KRASG12D mutated lung cancer. In addition, we investigated whether MRTX1133 effects the anti-tumor immunity in mice bearing lung cancer with KRASG12D. Method: CCSP-rtTA/TetO-Cre mice were crossed with LSL-KRASG12D knock-in mice to obtain CCSP-rtTA/TetO-Cre/LSL-KRASG12D conditional mutant mice (KRASG12D mice). These mice were fed an autoclaved rodent diet supplemented with 0.0625% doxycycline when the mice were about 8~12 weeks old. Mice were treated with 10 mg/kg MRTX1133 in vehicle once daily by intraperitoneal injection for 4 or 8 days. After administration of MRTX1133, the lungs and spleens were harvested, and single cell suspensions were labeled with fluorophore-conjugated antibodies for FACS analyses. Results: More regulatory T cells (Tregs) infiltrated the tumors of lung of mice treated with doxycycline for a longer period of time. The expression of immune checkpoint molecules (ICMs), including PD-L1, PD-1, TIGIT, TIM-3, LAG-3, were also upregulated on CD4+ and CD8+ T cells in the KRASG12D mice. Treatment with MRTX1133 strongly inhibited KRASG12D tumor progression, reduced the percentage of Tregs and downregulated the expression of PD-1 on CD4+ and CD8+ T cells infiltrating in lung cancers. The expression of the other ICMs remained high after MRTX1133 administration. In addition, MRTX1133 tended to increase the percentage of tumor-specific CD8+ effector T cells infiltration in lung tumors. Conclusion: T cells were exhausted in mice with KRASG12D mutant lung cancer. MRTX1133 demonstrates the therapeutic efficacy in preclinical murine KRASG12D mutated lung cancer model. This efficacy is more likely to relate not only signal inhibition but also enhances the anti- tumor immune responses. Our findings could aid in developing an alternative immunotherapy strategy in patients with KRASG12D mutant lung cancer. Citation Format: Kunihiro Shono, Satoshi Watanabe, Takaaki Masuda, Ryo Suzuki, Tomoya Wakabayashi, Tomoki Sekiya, Susumu Tanaka, Aya Ohtsubo, Tomohiro Tanaka, Koichiro Nozaki, Rie Kondo, Yu Saida, Satoshi Hokari, Naohiro Yanagimura, Masashi Arita, Riuko Ohashi, Kenjiro Shima, Yosuke Kimura, Nobumasa Aoki, Yasuyoshi Ohshima, Toshiyuki Koya, Toshiaki Kikuchi. The impact of MRTX1133 on anti-tumor immunity in lung cancer with KRASG12D mutation [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 5263.

Abstract 1468: Low exposures to amphibole or serpentine asbestos in germline Bap1-mutant mice induce mesotheliomacharacterized by an immunosuppressive tumor microenvironment

Abstract The purpose of this study was to determine if minimal exposures to amphibole (crocidolite) and serpentine (chrysotile) asbestos fibers significantly increase the incidence and rate of malignant mesothelioma (MM) onset in mice with a germline heterozygous Bap1 mutation, and to assess the effect of the resulting gene × environment interactions on the tumor immune microenvironment (TIME). The studies were carried out by chronically injecting intraperitoneally Bap1+/- and Bap1+/+ (wild-type, WT) mice with minimal doses of asbestos (in some experiments, as low as 0.0125 mg x 8 injections; total, 0.1 mg/mouse) and then monitoring the mice for tumor formation for up to 90 weeks. Pro-inflammatory and pro-tumorigenic factors in the TIME were evaluated by immunohistochemistry (IHC) and immunofluorescence (IF) for immune cell markers. With either type of asbestos and at each dose tested, MMs occurred at a significantly higher rate and earlier onset time in Bap1-mutant mice than in WT littermates. MMs from mice injected with chrysotile, but less so with crocidolite, had widespread adhesions in the peritoneal cavity owing to an overwhelming immune response, particularly at the higher dose used. IHC staining demonstrated an increased number of immune cells in granulomas and MMs. In particular, pro-inflammatory M1 macrophages were found mainly in granulomas, whereas pro-tumorigenic M2 macrophages were predominant in MMs. IF staining revealed that the relative number of M2 macrophages was strikingly greater in chrysotile-induced than crocidolite-induced MMs, suggesting that chrysotile induces a more profound immune response. MMs from Bap1-mutant mice showed upregulation of both CD39/CD73-adenosine and Ccl2/Ccr2 pathways, which collectively are known to mechanistically create favorable conditions for neoplastic transformation, invasion, and the development of an immunosuppressive TIME, in part by attracting M2 macrophages. This implies that chemoprevention and immunotherapy strategies targeting these pro-tumorigenic immune pathways could be beneficial in BAP1 mutation carriers. In conclusion, we provide evidence that low-level exposures to either chrysotile or crocidolite in germline Bap1 heterozygous mice result in a markedly increased risk of MM characterized by an immunosuppressive tumor microenvironment. Drawing parallels to humans, these experimental findings suggest that exposure of BAP1 mutation carriers to even minimal doses of different forms of asbestos may be associated with significantly enhanced susceptibility to MM. Citation Format: Yuwaraj Kadariya, Eleonora Sementino, Maggie Ruan, Mitchell Cheung, Andres J. Klein-Szanto, Kathy Cai, Joseph R. Testa. Low exposures to amphibole or serpentine asbestos in germline Bap1-mutant mice induce mesotheliomacharacterized by an immunosuppressive tumor microenvironment [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 1468.

Abstract 85: Immunomics of immune-deficient mouse models before and after CD34 humanization

Abstract Preclinical tumor oncology research relies historically on the analysis of mouse or human tumor cell lines implanted onto severely immune-deficient mouse models carrying nude, SCID or Rag mutations on different genetical background (C57Bl/6N, BalbC, NMRI, CB17 or NOD). These lines harbor defects in several leukocyte lineages among which Tc, Bc and NKc might be affected. Humanized mice are emerging models that have been transplanted with human cells or tissues (and/or equipped with human transgenes). Currently, the most advanced strains are the nonobese diabetic, severe combined immunodeficiency (NOD-SCID) mouse with complete disruptions in the interleukin-2 (IL-2) common γ-chain (IL2R γ null) receptor (NSG) and BALB/c Rag2−/− IL2R γ null SirpaNOD mice (BRGS).1 Neonate or juvenile chimera are reconstituted in NSG, BRGS or B6 RGS with CD34+ hematopoietic progenitor cell (HPCs) from human cord blood yielding robust engraftment of a human immune system (HIS).. We genetically modified the mouse genome introducing mutations involved in Tc, Bc and NKc development. We then immuno-phenotyped over 23 lines of mouse mutants produced from the same animal house to minimize the impact of microbiota on innate immune cell populations. High content cytometry analysis of several organs was performed in a standardized manner before and after humanization. The resource obtained of these immunotypes was used to highlight similarities and differences among these lines across several genetical background and will help in selecting the most appropriate model to use for tumor implantation and CD34 or PBMC humanization. Citation Format: Hervé Luche, Lilia Hadjem, Marielle Mello, Priscilla Canavese, Fabien Angelis, Anais Joachim, Sylvie Bouilly, Frédéric Guinut, Frederic Fiore, Bernard Malissen, Ana Zarubica, Erwan Corcuff. Immunomics of immune-deficient mouse models before and after CD34 humanization [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 85.

Mice with renal-specific alterations of stem cell-associated signaling develop symptoms of chronic kidney disease but surprisingly no tumors.

Previously, we found that Wnt and Notch signaling govern stem cells of clear cell kidney cancer (ccRCC) in patients. To mimic stem cell responses in the normal kidney in vitro in a marker-unbiased fashion, we have established tubular organoids (tubuloids) from total single adult mouse kidney epithelial cells in Matrigel and serum-free conditions. Deep proteomic and phosphoproteomic analyses revealed that tubuloids resembled renewal of adult kidney tubular epithelia, since tubuloid cells displayed activity of Wnt and Notch signaling, long-term proliferation and expression of markers of proximal and distal nephron lineages. In our wish to model stem cell-derived human ccRCC, we have generated two types of genetic double kidney mutants in mice: Wnt-β-catenin-GOF together with Notch-GOF and Wnt-β-catenin-GOF together with a most common alteration in ccRCC, Vhl-LOF. An inducible Pax8-rtTA-LC1-Cre was used to drive recombination specifically in adult kidney epithelial cells. We confirmed mutagenesis of β-catenin, Notch and Vhl alleles on DNA, protein and mRNA target gene levels. Surprisingly, we observed symptoms of chronic kidney disease (CKD) in mutant mice, but no increased proliferation and tumorigenesis. Thus, the responses of kidney stem cells in the tubuloid and genetic systems produced different phenotypes, i.e. enhanced renewal versus CKD.

Postnatal survival of phrenic motor neurons is promoted by BDNF/TrkB.FL signaling.

The number of motor neurons (MNs) declines precipitously during the final trimester before birth. Thereafter, the number of MNs remains relatively stable, with their connections to skeletal muscle dependent on neurotrophins, including brain-derived neurotrophic factor (BDNF) signaling through its high-affinity full-length tropomyosin-related kinase receptor subtype B (TrkB.FL) receptor. As a genetic knockout of BDNF leads to extensive MN loss and postnatal death within 1-2 days after birth, we tested the hypothesis that postnatal inhibition of BDNF/TrkB.FL signaling is important for postnatal phrenic MN (PhMN) survival. In the present study, we used a 1NMPP1-sensitive TrkBF616A mutant mouse to evaluate the effects of inhibition of TrkB kinase activity on phrenic MN (PhMN) numbers and diaphragm muscle (DIAm) fiber cross-sectional area (CSA). Pups were exposed to 1NMPP1 or vehicle (DMSO) from birth to 21 days old (weaning) via the mother's ingestion in the drinking water. Following weaning, the right phrenic nerve was exposed in the neck and the proximal end dipped in a rhodamine solution to retrogradely label PhMNs. After 24 h, the cervical spinal cord and DIAm were excised. Labeled PhMNs were imaged using confocal microscopy, whereas DIAm strips were frozen at ∼1.5× resting length, cryosectioned, and stained with hematoxylin and eosin to assess CSA. We observed an ∼34% reduction in PhMN numbers and increased primary dendrite numbers in 1NMPP1-treated TrkBF616A mice. The distribution of PhMN size (somal surface area) DIAm fiber cross-sectional areas did not differ. We conclude that survival of PhMNs during early postnatal development is sensitive to BDNF/TrkB.FL signaling.NEW & NOTEWORTHY During early postnatal development, BDNF/TrkB signaling promotes PhMN survival. Inhibition of BDNF/TrkB signaling in early postnatal development does not impact PhMN size. Inhibition of BDNF/TrkB signaling in early postnatal development does not impact the number or CSA of DIAm fibers.

CCDC157 is essential for sperm differentiation and shows oligoasthenoteratozoospermia-related mutations in men.

Oligoasthenoteratospermia (OAT), characterized by abnormally low sperm count, poor sperm motility, and abnormally high number of deformed spermatozoa, is an important cause of male infertility. Its genetic basis in many affected individuals remains unknown. Here, we found that CCDC157 variants are associated with OAT. In two cohorts, a 21-bp (g.30768132_30768152del21) and/or 24-bp (g.30772543_30772566del24) deletion of CCDC157 were identified in five sporadic OAT patients, and 2 cases within one pedigree. In a mouse model, loss of Ccdc157 led to male sterility with OAT-like phenotypes. Electron microscopy revealed misstructured acrosome and abnormal head-tail coupling apparatus in the sperm of Ccdc157-null mice. Comparative transcriptome analysis showed that the Ccdc157 mutation alters the expressions of genes involved in cell migration/motility and Golgi components. Abnormal Golgi apparatus and decreased expressions of genes involved in acrosome formation and lipid metabolism were detected in Ccdc157-deprived mouse germ cells. Interestingly, we attempted to treat infertile patients and Ccdc157 mutant mice with a Chinese medicine, Huangjin Zanyu, which improved the fertility in one patient and most mice that carried the heterozygous mutation in CCDC157. Healthy offspring were produced. Our study reveals CCDC157 is essential for sperm maturation and may serve as a marker for diagnosis of OAT.

Stem cell factor and cKIT modulate endothelial glycolysis in hypoxia.

In hypoxia, endothelial cells (ECs) proliferate, migrate, and form new vasculature in a process called angiogenesis. Recent studies have suggested that ECs rely on glycolysis to meet metabolic needs for angiogenesis in ischaemic tissues, and several studies have investigated the molecular mechanisms integrating angiogenesis and endothelial metabolism. Here, we investigated the role of stem cell factor (SCF) and its receptor, cKIT, in regulating endothelial glycolysis during hypoxia-driven angiogenesis. SCF and cKIT signalling increased the glucose uptake, lactate production, and glycolysis in human ECs under hypoxia. Mechanistically, SCF and cKIT signalling enhanced the expression of genes encoding glucose transporter 1 (GLUT1) and glycolytic enzymes via Akt- and ERK1/2-dependent increased translation of hypoxia inducible factor 1A (HIF1A). In hypoxic conditions, reduction of glycolysis and HIF-1α expression using chemical inhibitors significantly reduced the SCF-induced in vitro angiogenesis in human ECs. Compared with normal mice, mice with oxygen-induced retinopathy (OIR), characterized by ischaemia-driven pathological retinal neovascularization, displayed increased levels of SCF, cKIT, HIF-1α, GLUT1, and glycolytic enzymes in the retina. Moreover, cKIT-positive neovessels in the retina of mice with OIR showed elevated expression of GLUT1 and glycolytic enzymes. Further, blocking SCF and cKIT signalling using anti-SCF neutralizing IgG and cKIT mutant mice significantly reduced the expression of HIF-1α, GLUT1, and glycolytic enzymes and decreased the pathological neovascularization in the retina of mice with OIR. We demonstrated that SCF and cKIT signalling regulate angiogenesis by controlling endothelial glycolysis in hypoxia and elucidated the SCF/cKIT/HIF-1α axis as a novel metabolic regulation pathway during hypoxia-driven pathological angiogenesis.

Photoreceptor deficits appear at eye opening in Rs1 mutant mouse models of X-linked retinoschisis

X-linked retinoschisis (XLRS) is an early onset degenerative retinal disease characterized by cystic lesions in the middle layers of the retina. These structural changes are accompanied by a loss of visual acuity and decreased contrast sensitivity. XLRS is caused by mutations in the gene Rs1 which encodes the secreted protein Retinoschisin 1. Young Rs1-mutant mouse models develop key hallmarks of XLRS including intraretinal schisis and abnormal electroretinograms. The electroretinogram (ERG) comprises activity of multiple cellular generators, and it is not known how and when each of these is impacted in Rs1 mutant mice. Here we use an ex vivo ERG system and pharmacological blockade to determine how ERG components generated by photoreceptors, ON-bipolar, and Müller glial cells are impacted in Rs1 mutants and to determine the time course of these changes. We report that ERG abnormalities begin near eye-opening and that all ERG components are involved.

Hybrid Molecular and Functional Micro-CT Imaging Reveals Increased Myocardial Apoptosis Preceding Cardiac Failure in Progeroid Ercc1 Mice

PurposeIn this study, we explored the role of apoptosis as a potential biomarker for cardiac failure using functional micro-CT and fluorescence molecular tomography (FMT) imaging techniques in Ercc1 mutant mice. Ercc1 is involved in multiple DNA repair pathways, and its mutations contribute to accelerated aging phenotypes in both humans and mice, due to the accumulation of DNA lesions that impair vital DNA functions. We previously found that systemic mutations and cardiomyocyte-restricted deletion of Ercc1 in mice results in left ventricular (LV) dysfunction at older age.Procedures and ResultsHere we report that combined functional micro-CT and FMT imaging allowed us to detect apoptosis in systemic Ercc1 mutant mice prior to the development of overt LV dysfunction, suggesting its potential as an early indicator and contributing factor of cardiac impairment. The detection of apoptosis in vivo was feasible as early as 12 weeks of age, even when global LV function appeared normal, underscoring the potential of apoptosis as an early predictor of LV dysfunction, which subsequently manifested at 24 weeks.ConclusionsThis study highlights the utility of combined functional micro-CT and FMT imaging in assessing cardiac function and detecting apoptosis, providing valuable insights into the potential of apoptosis as an early biomarker for cardiac failure.