Emerging evidence shows that Proton FLASH radiotherapy can spare normal tissues while maintaining anti-tumor efficacy. However, its impact on intestinal stem cells (ISCs) and the gut microbiome remains unclear. Gut microbiome influences ISC's radiosensitivity. In a mouse model of abdominal irradiation, Proton FLASH exposure exhibited improved survival and less crypt-villus damage compared to Proton Conventional dose rate. Using scRNA-sequencing, we demonstrated that Proton FLASH exposure using pulsed pencil beam scanning spares two distinct ISC populations, Lgr5+ Crypt-based columnar cells (CBCs) and a Ly6a+, Clu+, Areg+, Anxa2+ revival stem cell (revSC) population-by modulating oxidative stress and cell cycle progression. Analysis of α and β-diversity demonstrated that Proton FLASH modulates gut microbiota composition without compromising overall species richness. Notably, Proton FLASH-irradiated mice had higher abundances of Alistipes sp. and Akkermensia sp., both known for protective effects on ISCs and the intestinal mucosa. The role of microbiome in Proton FLASH-mediated sparing effect was further confirmed by fecal microbiota transplantation, where Proton FLASH-donor microbiota demonstrated reduced lethality with protection of crypt villus morphology in recipient mice exposed to Proton Conventional dose rate. Our findings highlight the crucial role of the microbiome in the Proton FLASH-mediated sparing of the mucosal epithelium.

Background:Familial adenomatous polyposis (FAP) is an inherited predisposition to colorectal cancer and characterized by profuse colorectal adenomas starting from the second decade of life. Regional (left vs. right) differences in the colonic microbiologic and immunologic microenvironment may impact adenoma evolution but are poorly understood. We aimed to characterize regional molecular, microbial, DNA damage, and immune differences in pediatric FAP polyps to test the hypothesis that polyps in pediatric FAP exhibit distinct regional and molecular features that contribute to differential growth and genomic instability. Methods:Colonic polyps and adjacent non-polyp mucosa were harvested from pediatric FAP patients undergoing colonoscopy. Tandem mass tag-based proteomic and phosphoproteomic profiling was performed and were followed by functional assays including colony formation, spheroid growth, and patient-derived organoid culture. γH2AX staining was used to quantify induction of DNA double-strand breaks (DSBs) in HCT116 colon cancer cells cultured in Fusobacterium nucleatum conditioned media (FnCM). Immunohistochemistry and immunofluorescence were used to assess ATR, CDK4, γH2AX, and oxidative damage (8-OxoG). Immune profiling was performed by flow cytometry, focusing on CD103+ tissue-resident memory T cells (TRMs). Results:Right-sided polyps exhibited increased ATR and CDK4 expression compared with left-sided lesions and adjacent mucosa. FnCM exposure induced a marked increase in γH2AX staining in HCT116 cells, consistent with our in vivo findings of elevated DSB burden in proximal versus distal FAP polyps. Biofilm enrichment and higher microbial staining were observed in right-sided lesions, whereas distal polyps were enriched with CD103+ TRM populations. Pharmacologic inhibition of ATR or CDK4 significantly suppressed both colony formation and spheroid growth. Organoids derived from proximal colon polyps exhibited accelerated growth and crypt budding, with higher expression of stemness markers (CD44, CD133, Lgr5, BMI-1) compared with distal polyps. Conclusions:Integrated proteomic, phosphoproteomic, and immune-microbiome profiling reveals regional heterogeneity of adenomas in pediatric FAP. Right compared to left sided polyps harbor greater DNA damage, elevated ATR/CDK4 kinase activity, reduced immune surveillance, and increased stem-like growth. These findings identify ATR and CDK4 as potential therapeutic targets and suggest that regional microenvironmental differences can impact chemoprevention strategies in pediatric FAP.

Abstract Background: The failure of current DNA damage response strategies to adequately distinguish tumor from normal tissue remains a major barrier to durable cancer control. BCN077 is a first-in-class small-molecule activator of human single strand DNA-binding protein 2 (hSSB2), a conserved single-stranded DNA binding complex that coordinates replication stress responses, DNA repair, and checkpoint recovery. We hypothesized that pharmacologic activation of hSSB2 would create a therapeutically exploitable divergence: lethal replication stress in genomically unstable tumors, but epithelial protection in normal tissues exposed to radiation or chemotherapy. Methods: BCN077 was evaluated across cancer cell lines, the NCI-60 panel, and in vivo tumor and radiation injury models. Mechanistic studies assessed DNA damage, replication stress, spindle checkpoint signaling, and mitotic catastrophe using γH2AX, phospho-H3, BUB1, MAD2L1, comet analysis, clonogenic survival, and morphologic criteria. Tumor efficacy was tested in aggressive and treatment-resistant models, including BRAF V600E colorectal cancer, and normal tissue protection was assessed in irradiation settings relevant to gastrointestinal injury. Results: BCN077 demonstrated broad anti-tumor activity across diverse cancer types and selectively triggered mitotic catastrophe in checkpoint-defective tumor cells characterized by unresolved DNA damage, aberrant mitotic entry, and loss of proliferative capacity. In contrast, normal cells with intact checkpoint function were comparatively spared. In vivo, BCN077 enhanced tumor control and showed marked protective activity in irradiated or chemo exposed normal epithelium, preserving intestinal architecture and improving survival after radiation exposure. These findings support a mechanism in which hSSB2 activation intensifies genotoxic stress beyond the tolerable threshold in cancer cells while reinforcing recovery and survival programs in normal tissues. Conclusions: hSSB2 is an emerging druggable vulnerability with a compelling dual therapeutic profile. BCN077 may define a new class of agents that simultaneously improve tumor control and reduce treatment-limiting toxicity, representing a genuine translational opportunity to expand the therapeutic window of radiation and chemotherapy. This strategy is especially relevant for malignancies in which checkpoint failure and replication stress are common, including triple-negative breast cancer not amenable to PARP inhibition and head and neck cancers where improved tumor ablation must be balanced against epithelial sparing. Citation Format: Andrew J. Norris, Elizabeth M. Singer, Rishi Man Chugh, Payel Bhanja, Julian P. Whitelegge, William H. McBride, Jesus M. Rodriguez, Natalie Isaghulian, Alexander M. Varady, Subhrajit Saha. Activation of hSSB2/1 by BCN077 Induces Tumor-Selective Mitotic Catastrophe While Protecting Irradiated Epithelium: A New Therapeutic Paradigm for Expanding the Cancer Treatment Window [abstract]. In: Proceedings of AACR Drug Discovery and Development (AACR D3) Conference; 2026 Jul 21-24; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(14_Suppl):Abstract nr A080.

Introduction:Pancreatic adenocarcinoma (PDAC) is an aggressive malignancy with a poor prognosis. While germline mutations in BRCA1/2 are well-established risk factors, mutations in the BLM gene (associated with Bloom Syndrome) are rare in this context. Case Presentation:We present a 73-year-old female with a recent history of small cell lung cancer (SCLC) who presented with metastatic pancreatic adenocarcinoma. Genetic profiling revealed a pathogenic germline BLM mutation and a somatic ATM mutation. The patient was treated with an oxaliplatin-based regimen (mFOLFOX6), modified due to comorbidities, achieving disease stabilization. Discussion:This case highlights the complexity of managing metachronous malignancies and the utility of comprehensive genomic profiling. The presence of pathogenic variants in DNA damage response (DDR) genes (BLM and ATM) suggests a defect in homologous recombination, providing a rationale for platinum-based therapy. We discuss the implications of BLM mutations on therapeutic selection, potential immune checkpoint interactions, and the role of synthetic lethality in management.

e16484 Background: Pancreatic acinar cell carcinoma (PACC) is an ultra-rare (~1%) pancreatic malignancy. Management is often extrapolated from pancreatic ductal adenocarcinoma (PDAC) despite distinct molecular drivers. We characterize the clinicopathologic and genomic features of PACC at our center to identify precision oncology targets. Methods: A retrospective review of histologically confirmed PACC at a tertiary academic center was performed. Clinical, radiographic, pathologic, treatment, and outcome data were extracted. Standard of care next-generation sequencing (NGS) results were reviewed, with focus on potentially actionable alterations. Results: Seven patients were identified (median age 64, range 46–70; 57% male). Symptoms included abdominal pain and weight loss; notably, jaundice was absent. Pathologic evaluation demonstrated variable lymph node involvement, lymphovascular and perineural invasion, and occasional mixed acinar neuroendocrine differentiation. NGS was performed in a subset of patients and actionable alterations included 9p21.3 co-deletion (CDKN2A/B and MTAP loss), IZKF1 loss, SEC24D-BRAF fusion, and BAP1 mutation (Table 1). All were KRAS wild-type. Surgical resection was performed in three patients, and FOLFIRINOX chemotherapy was commonly used. Treatment responses varied. A swimmer plot analysis revealed that 2/7 patients had prolonged progression-free survival ( > 90months and > 200months), while the remaining patients had follow-up < 5 years. At the time of data reporting, four patients were alive without disease recurrence and under ongoing surveillance. Conclusions: Our cohort confirms PACC is molecularly distinct from PDAC, characterized by absence of KRAS mutation and high prevalence of targetable alterations. The identification of MTAP loss and BRAF alteration suggests that PACC patients should be prioritized for specific precision medicine trials (e.g., PRMT5 or MAPK inhibitors) rather than traditional PDAC regimens. Early NGS is mandatory to optimize precision treatment in this rare cancer. Patient Age Sex Presenting Symptoms Tumor Location Resection performed NGS Findings MSI Status TMB Potential Target 1 46 F Abdominal pain, nausea, bloating, fatigue Head Yes Not tested Not tested Not tested N/A 2 68 F Pruritis, abdominal pain, appetite loss, constipation/diarrhea Head Yes TP53 mutation; KRAS wild type; CDKN2A/B loss; MTAP loss; SEC24D-BRAF fusion MSS Intermediate BRAF/MEK/PRMT5 inhibitors 3 67 M Abdominal pain Head Attempted (aborted) Not tested Not tested Not tested N/A 4 70 M Flank pain and hematuria Head No Not tested Not tested Not tested N/A 5 86 F Asymptomatic (incidental lab finding) Head No BAP1 mutation MSS 6.6 EZH2 inhibitors 6 55 M Abdominal pain Tail No CDKN2 A/B loss; IKZF1 loss; MTAP loss; KRAS wild type MSS 6.8 PRMT5 inhibitors 7 57 M Asymptomatic (incidental imaging finding) Head Yes Not tested Not tested Not tested N/A

Abstract Pancreatic cancer patients frequently undergo radiotherapy, which carries a significant risk of toxicity to the small bowel and gastrointestinal tissues. Therapeutic outcomes in pancreatic cancer often depend on delivering higher radiation doses, and thus strategies that minimize gastrointestinal toxicity without compromising tumor radiosensitivity would be profoundly beneficial to patients. Radiation induced oxidative stress plays a significant role in radiation induced toxicity in intestinal epithelial cells. However, mitigation of gastrointestinal toxicity requires reduction of oxidative stress and stimulation of epithelial regeneration through activation of WNT/β-catenin signaling. Mitochondrial serine/threonine phosphatase, phosphoglycerate mutase 5 (PGAM5), is involved in activation of oxidative stress along with inhibition of WNT/β-catenin signaling. PGAM5, located in the mitochondrial membrane, inhibits nuclear translocation of NRF2 and thereby represses NRF2-dependent antioxidant gene expression. Moreover, PGAM5 induces β-catenin degradation by dephosphorylating DVL2, a β-catenin destruction complex. We have observed PGAM5 expression significantly increased in intestinal epithelium in response to irradiation. Pharmacological inhibition of PGAM5 using a novel small molecule-based modulator LFHP-1c (3 mg/kg BW, subcutaneous) at 24 hours post irradiation mitigates gastrointestinal toxicity. Mice exposed to partial body irradiation (PBI) with 2.5% bone marrow shielding (LD100/15) followed by LFHP-1c treatment demonstrated significant improvement of mice survival (90% mice survived beyond 30 day) (p< 0.00002) compared to irradiated control where all the mice died within 14 days. Histopathological analyses demonstrated preservation of crypt-villus structures in the jejunum sections of LFHP-1c-treated mice compared to untreated irradiated mice. LFHP-1c treatment significantly (p<0.005) induced stabilization and nuclear translocation of NRF2, along with increased nuclear localization of β-catenin, indicating activation of the WNT/β-catenin signaling pathway in the irradiated intestinal epithelium. LFHP-1c treatment in irradiated organoids from Lgr5/eGFP-IRES-Cre-ERT2; R26-ACTB-tdTomato-EGFP mice intestine demonstrated mitigation of radiation induced toxicity and significant improvement in Lgr5+ve intestinal stem cell survival. In mice model of Pancreatic tumors, LFHP-1c treatment did not compromise the radiosensitivity of Kras positive KPC cells. In conclusion, our studies using mice model of radiation induced genotoxic stress and ex vivo organoid model demonstrated that PGAM5 can be a potential target to promote therapeutic ratio for abdominal radiotherapy. Citation Format: Shujah Hamid Rehman, Rishi Man Chugh, Payel Bhanja, Stacey Krepel, Subhrajit Saha, . Mitochondrial serine/threonine phosphatase, phosphoglycerate mutase 5 (PGAM5) is a novel target to promote abdominal radiotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4628.

Abstract BCN077 is a first-in-class small-molecule activator of the human single-stranded DNA binding complex hSSB2/1, a key regulator of replication stress responses and DNA repair. In cancer cells with high baseline replication stress and defective cell-cycle checkpoints, pharmacologic activation of hSSB2/1 by BCN077 drives excessive replication fork processing and accumulation of unrepaired double-strand breaks, forcing damaged cells through mitosis and triggering mitotic catastrophe. In contrast, normal stem and progenitor cells with intact checkpoints use hSSB2/1 activation to pause, repair therapy-induced DNA damage more efficiently, and survive, creating a therapeutically favorable differential between tumor killing and normal tissue protection. This dual mechanism positions BCN077 represents a novel DNA damage–response drug class that couples synthetic lethality in genomically unstable tumors with concurrent radio/chemo-protection of normal tissues. In a BRAFV600E colorectal cancer model using RKO xenografts in male athymic BALB/c nu/nu mice, we are evaluating the novel agent BCN077 in both naïve and treatment-resistant settings. RKO typically exhibits immunosuppressive microenvironments with reduced T-cell presence and high myeloid-derived suppressor cell activity. Mice were randomized to four arms: vehicle control, BCN077 monotherapy, standard FOLFOX plus Encorafenib (Braftovi®) and cetuximab, or the same triplet regimen combined with BCN077. Further we have evaluated the effect of BCN077 with respect to method of cell death confirming mitotic catastrophe as the form of cell death not only in RKO but also Panc-1 and other cancer cell lines showing positive Histone H-3, gamma H2AX stain, comet assay Bub1 and MAD1 staining among other key biomarkers. The NCI-60 panel also was conducted across cancer types showing effectiveness across cancer types with growth inhibition (GI50), Lethal concentration (LC50) and total growth inhibition (TGI) data. The normal cell line HEK293 did not show positive under the same conditions having intact cell cycle checkpoint control. Lastly, in vivo data clearly indicate that BCN077 has profound protective effects from cytotoxic therapy employed in cancer treatment such as radiation. hSSB2 is a novel target and holds great promise for use in cancer therapy, in particular where there is resistance to targeted therapeutics in the MAPK pathway such as RAS, BRAF, MEK inhibitors where resistance enables cell cycle checkpoint evasion. Citation Format: Elizabeth M. Singer, Rishi M. Chugh, Payel Bhanja, Julian P. Whitelegge, William H. McBride, Jesus Rodriguez, Anusha Ravisankar, Mandeep Kumari, Andrew John Norris, Subhrajit Saha. First in class hSSB2 activator as a dual therapeutic strategy for overcoming RAS/MAPK inhibitor resistance while protecting normal epithelia in aggressive cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr LB352.

Abstract Our study was done to elucidate the mechanism of sex-dependent differences in radiotherapy (RT) response in males versus females, and then utilize this mechanism to help prevent intestinal radiation toxicity. More than 50% of patients with gastrointestinal (GI) cancers undergo abdominal radiotherapy. However, intestinal epithelial radiosensitivity is a major limiting factor to delivering a tumoricidal dose. Personalized differences, including sex-specific differences in radiosensitivity, is one of the key determining factors in radiotherapy outcome. Using a mouse model of abdominal irradiation and a human intestinal organoid model, we previously demonstrated that healthy male intestinal stem cells are more radiosensitive than females due to higher rates of oxidative phosphorylation (OXPHOS) and production of reactive oxidative species (ROS). In the present study, we demonstrate that these higher rates of OXPHOS in males are due to increased expression of the Mitochondrial Pyruvate Carrier (MPC), which transports pyruvate into the mitochondria for flux through the TCA cycle, and, ultimately, the OXPHOS pathway. Genetic deletion of the MPC in Lgr5-EGFP-positive ISCs increases ISC survival following the reduction in radiation-induced mitochondrial pyruvate oxidation in both male and female organoids. In both human intestinal organoids and a mouse model of radiation-induced gastrointestinal syndrome, treatment with MPC inhibitor, UK5099, normalized these differences in radiation responses between males and females. Moreover, our study in a mouse model of pancreatic adenocarcinoma also establishes UK5099 as a radio-modulator for pancreatic cancer, as combination of RT+ UK5099 treatment significantly reduces tumor growth and alters the immunosuppressive tumor immune microenvironment compared to irradiated control. These findings clearly suggest that pyruvate metabolism and MPC can be a potential target to promote therapeutic ratio of abdominal radiotherapy. Citation Format: Stacey Krepel, Payel Bhanja, Rishi Man Chugh, Shujah Hamid Rehman, Subhrajit Saha. Reprogramming of pyruvate metabolism overcomes sex-specific differences in intestinal stem cell radiosensitivity and improves the therapeutic ratio for abdominal irradiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5267.

Prolonged colonic inflammation and ulcerative colitis lead to colon cancer. The rapid growth and treatment-resistant nature of these tumors are primarily influenced by an immunosuppressive tumor microenvironment, which is led by tumor-associated macrophages (TAMs). However, factors influencing or regulating the immunosuppressive nature of TAMs have not been sufficiently studied. In this manuscript, we use a mouse model of colitis-associated colorectal cancer (CRC) to demonstrate that WNT expression in TAMs regulates their immunosuppressive function by inhibiting Glycogen synthase kinase-3 beta (GSK-3β) within the macrophages, possibly through an autofeedback loop. GSK3β is a positive regulator of PD-1 and PDL1 expression in macrophages and promotes an immunosuppressive microenvironment. Therefore, GSK-3β inhibition alters the immunosuppressive nature of the immune microenvironment and effectively controls tumor growth. In Csf1r-iCre; Porcnfl/fl mice, the absence of macrophage-derived WNT promotes tumor growth in the model of colitis-associated colon cancer. Absence of macrophage-derived WNT stabilizes GSK-3β in macrophages and promotes an immunosuppressive tumor microenvironment. We also show that pharmacological inhibition of GSK-3β in a macrophage-specific manner, achieved by systemic delivery of a lipo-GSK3β inhibitor, effectively inhibits tumor growth. Therefore, this manuscript demonstrates for the first time that the macrophage-specific modulation of GSK3β can be a potential target to treat colitis-associated colon cancer. ### Competing Interest Statement The authors have declared no competing interest.

Macrophages are the major source of WNT ligands. However, the regulation of WNT expression in macrophages has not been studied. In the present study, we have discovered that activation of canonical β-Catenin signaling suppresses WNT expression in macrophages. EVs from these pre-conditioned macrophages promoted intestinal stem cell regeneration and mitigated intestinal injury. ChIP-seq analysis and validation studies using recombinant DNA construct expressing Luciferase reporter under WNT promoter (e.g. WNT5a and WNT9b) were conducted to demonstrate the involvement of β-Catenin in the transcriptional regulation of WNT expression. The regulatory role of β-Catenin in WNT expression in macrophages was examined by treating these cells with a Tankyrase inhibitor. In addition, the gene expressing β-Catenin was deleted in macrophages using Csf1r.iCre; Ctnnb1fl/fl mice model. Both pharmacological and genetically modulated macrophages were examined for WNT expression and activity by qPCR and TCF/LEF luciferase assay respectively. Additionally, Csf1r.iCre; Ctnnb1fl/fl mice were exposed to irradiation to compare the radiosensitivity with their wildtype littermate. Extracellular vesicles (EVs) were isolated from pre-conditioned WNT-enriched macrophages and infused in irradiated C57BL/6 and Lgr5/eGFP-IRES-Cre-ERT2; R26-ACTB-tdTomato-EGFP mice to determine the regenerative response of intestinal stem cell (ISC) and epithelial repair. Regenerative effects of EVs were also examined in mice model DSS induced colitis. ChIP-seq analysis and subsequent validation study suggested physical association of β-Catenin with WNT promoters to suppress WNT expression. Macrophage specific deletion of gene expressing β-Catenin or pharmacological inhibition of Tankyrase improves the WNT expression in macrophages several folds compared to control. Transfusion of these preconditioned macrophages or EVs from these cells delivers optimum level of morphogenic WNT to injured epithelium, activates ISC regeneration and mitigated radiation induced intestinal injury. Intestinal epithelium in Csf1r.iCre; Ctnnb1fl/fl mice also showed radioresistance compared to wild type littermate. Moreover, EVs derived from WNT enriched macrophages can mitigate intestinal injury in mice model of DSS induced acute colitis. The study provides substantial evidence that macrophage-targeted modulation of canonical WNT signaling induces WNT expression in macrophages. Treatment with preconditioned macrophage derived WNT-enriched EVs can be a promising therapeutic approach against intestinal injury.

Certepetide (aka CEND-1, LSTA1) is a tumor-penetrating peptide that binds integrin αvβ3 on tumor endothelium and neuropilin-1, triggering transcytosis to enhance intratumoral drug delivery and modulate the tumor microenvironment (TME). We report findings from resectable and borderline resectable PDAC of the CENDIFOX trial evaluating Certepetide plus mFOLFIRINOX as neoadjuvant therapy. Eligible patients with resectable and borderline resectable PDAC received neoadjuvant mFOLFIRINOX for 3 cycles followed by addition of Certepetide (3.2 mg/kg IV on Day 1) to mFOLFIRINOX every 2 weeks from cycles 4 onward for at least 6 cycles, followed by evaluation for resection. The primary objective was safety; secondary endpoints included resection rate, pathologic response, PFS, OS, and immune profiling. Correlative biopsies were obtained pre-treatment and at end of therapy. 35 patients were enrolled. No dose-limiting toxicities were observed. Common Grade ≥3 AEs included neutropenia, mucositis, fatigue, anorexia, and gastrointestinal events. Toxicities were manageable with dose reductions or delays. Most AEs were attributed to mFOLFIRINOX; no serious AEs were attributed to Certepetide. Of the 35 patients enrolled, 10 underwent pancreatic cancer resection following treatment regimen. Among these evaluable cases, the pathologic partial response rate (Tumor Regression Grade 2) was 70%, and the R0 resection rate was 50%. At limited follow-up, the 2-year OS rate was 60% (95% CI, 26%–100%), and median DFS was 12 months (95% CI, 10–NA). Immunofluorescence staining of PDAC tissue demonstrated increased post-treatment expression of CD68 (tumor-associated macrophages, TAMs) and immune checkpoints PD-1/PD-L1. Mean log-transformed CD68 intensity increased from 13.96 to 15.20; PD-1 from 12.94 to 13.57; and PD-L1 from 13.33 to 13.54, suggesting enhanced immune infiltration. Certepetide combined with mFOLFIRINOX is safe and feasible in resectable PDAC. Encouraging early OS and PFS data, high pathologic partial response rates, and correlative immune findings support further evaluation in randomized trials. Enhancement of TAMs and PD-1/PD-L1 in the tumor microenvironment supports the potential to convert PDAC from an immune-cold to an immune-hot tumor, possibly sensitizing it to immunotherapy. NCT05121038 Anup Kasi, Raed Al-Rajabi, Anwaar Saeed, Jianzheng Wu, Milind Phadnis, Shannon Bradbury, Stacey Krepel, Subhrajit Saha, Grace Li Haug, Prasad Dandawate, Rashna Madan, Mojtaba Olyaee, Amit Rastogi, Timothy Schmitt, Sean Kumer, Weijing Sun, Joaquina Baranda. CENDIFOX: Phase I/II Trial of CEND-1 (LSTA1, certepetide) with Neoadjuvant mFOLFIRINOX in Resectable and Borderline Resectable PDAC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research—Emerging Science Driving Transformative Solutions; Boston, MA; 2025 Sep 28-Oct 1; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_3):Abstract nr A070.

Macrophages are essential for maintaining tissue homeostasis and accelerating the repair processes; however, their functionality can be severely compromised in pathological conditions such as radiation-induced dermatitis. In this study we analyzed the role of macrophage derived Vascular Endothelial Growth Factor (VEGF) on regulation of macrophage senescence and its role on radiation-induced skin damage. We used bone marrow-derived macrophages (BMMɸ) isolated from Csf1r-iCre; VEGFfl/fl (VEGF-null) and wild-type (WT) mice. Macrophages were exposed to oxidative and genotoxic stress using H₂O₂, doxorubicin, and radiation exposure to evaluate senescence. Senescence was assessed via SA-β-Gal staining and expression of senescence-related genes. Additionally, VEGF receptor inhibition in WT macrophages was performed to determine the role of VEGF/VEGFR signaling in senescence regulation. Phagocytosis and migration assays were conducted to evaluate functional differences. For in vivo analysis, WT and Csf1r-iCre; VEGFfl/fl mice were exposed to radiation, and skin toxicity, histological changes, and senescence markers in skin macrophages were assessed. VEGF-null macrophages showed increased sensitivity to senescence, with elevated SA-β-Gal staining and upregulated senescence-associated gene expression. WT macrophages treated with a VEGF receptor inhibitor displayed increased senescence-associated markers expression, highlighting the importance of VEGF/VEGF-R signaling in preventing macrophage senescence-like phenotypes. Additionally, VEGF-null macrophages have reduced phagocytic and migratory abilities. Our in vivo study using Csf1r-iCre; VEGFfl/fl mice showed more severe radiation-induced dermatitis, including increased skin toxicity, hyperkeratosis, and elevated senescence-associated markers in skin macrophages compared to WT controls. Absence of macrophage-derived VEGF leads to heightened macrophage dysfunction and exacerbates radiation-induced dermatitis. Targeting VEGF signaling may serve as a potential therapeutic strategy to mitigate radiation-related skin toxicity and improve patient outcomes during radiation therapy.

Macrophages are the major source of WNT ligands. Macrophage-derived WNT is one of the most potent regenerative signals to mitigate intestinal injury. However, regulation of WNT expression in macrophages has not been studied. In the present study, we discovered that activation of canonical β-Catenin suppresses WNT expression in macrophages. Our CHIP-seq and validation study demonstrated the involvement of β-Catenin in the transcriptional regulation of WNT expression. Genetic and pharmacological approaches to de-stabilize/inactivate β-Catenin induce WNT expression in macrophages. Extracellular vesicles (EVs) are a major career of WNT ligands. Transfusion of EVs from pre-conditioned WNT-enriched macrophages demonstrated significant regenerative benefit over native macrophage-derived EVs to mitigate radiation-induced intestinal injury. Transfusion of WNT-enriched EVs also reduces DSS-induced colitis. Our study provides substantial evidence to consider that macrophage-targeted modulation of canonical WNT signaling to induce WNT expression followed by treatment with WNT-enriched EVs can be a lead therapy against intestinal injury.. SUMMARY:Activation of β-Catenin suppresses WNT expression in macrophages. Macrophage-targeted pharmacological modulation of canonical WNT signaling followed by adoptive transfer mitigate radiation injury in intestine. EVs from these preconditioned macrophages mitigate chemical or radiation induced intestinal injury.

Tissue radiosensitivity plays a critical role in the overall outcome of radiation therapy. Identifying characteristics that predict how a patient may respond to radiotherapy enables clinicians to maximize the therapeutic window. Limited clinical data have suggested a difference in male and female radiotherapy outcomes. Radiotherapy for gastrointestinal malignancy is still a challenge due to intestinal sensitivity to radiation toxicity. In this manuscript, we demonstrated sex-specific differences in intestinal epithelial radiosensitivity. In a mouse model of abdominal irradiation, we observed a significant increase in oxidative stress and injury in males compared to females. Lgr5+ve intestinal stem cells from male mice showed higher sensitivity to radiation-induced toxicity. However, sex-specific differences in intestinal radiosensitivity were not dependent on sex hormones, as we demonstrated similar sex-specific radiosensitivity differences in pre-pubescent mice. In an ex vivo study, we found that patient-derived intestinal organoid (PID) from males showed higher sensitivity to radiation compared to females as evident from loss of budding crypts, organoid size, and membrane integrity. Transcriptomic analysis of human Lgr5+ intestinal stem cells suggested radiation-induced upregulation of mitochondrial oxidative metabolism in males compared to females, a possible mechanism for radiosensitivity differences.

Radiosensitivity, the susceptibility of cells to ionizing radiation, plays a critical role in understanding the effects of radiation therapy and exposure on tissue health and regeneration. Identifying characteristics that predict how a patient may respond to radiotherapy enables clinicians to maximize the therapeutic window. Limited clinical data suggested a difference in male and female radiotherapy outcomes. Radiotherapy for gastrointestinal malignancy is still a challenge due to intestinal sensitivity to radiation toxicity. In this manuscript, we demonstrated sex-specific differences in intestinal epithelial radiosensitivity. In mice models of abdominal irradiation, we observed a significant increase in oxidative stress and injury in males compared to females. Lgr5+ve intestinal stem cells from male mice showed higher sensitivity to radiation-induced toxicity. However, sex-specific differences in intestinal radiosensitivity are not dependent on sex hormones as we demonstrated similar sex-specific radiosensitivity differences in pediatric mice. In an ex-vivo study, we found that human patient-derived intestinal organoids (PID) derived from males showed higher sensitivity to irradiation compared to females as evidenced by loss of budding crypt, organoid size, and membrane integrity. Transcriptomic analysis of human Lgr5+ intestinal stem cells suggested radiation induced upregulation of mitochondrial oxidative metabolism in males compared to females' possible mechanism for radiosensitivity differences.

mRNA expression of UPR downstream proapoptotic molecule