Abstract The tumor suppressor gene TP53 is frequently mutated in most solid malignancies, including colorectal and pancreatic cancers, driving tumor progression and metastasis. However, existing treatments often lack selectivity for p53-mutant (p53mut) cancers and are associated with high toxicity. To address this clinical challenge, we developed a two drug therapeutic approach that selectively targets p53-mutant cancers by combining TAS102, with a PARP inhibitor (PARPi). Mechanistically, incorporation of TFT into DNA triggers post-replicative repair, generating single-strand break intermediates. While PARP facilitates their repair, inhibition of PARP converts these intermediates into lethal double-strand breaks. In p53 wild-type (WT) cells, TAS102 and PARPi activate a p53-dependent G1/S checkpoint, enabling DNA repair and preventing excessive damage. In contrast, p53mut cells, lacking this checkpoint, experience uncontrolled DNA damage accumulation, leading to cell death. This combination demonstrated superior anti-tumor efficacy in p53-mutant cell lines and patient-derived xenograft (PDX) models compared to either agent alone, and was well tolerated in preclinical studies. This two-drug strategy is now being tested in Phase I clinical trial (NCT04511039) in advanced CRC patients showed no significant toxicity and improved PFS relative to historical TAS102 monotherapy. To further elucidate its mechanism, we investigated the DNA damage response (DDR) and checkpoint signaling in p53mut cancer cells. Our findings show that TAS102-PARPi induces a p53-independent G2/M checkpoint mediated by ATR kinase, which activates downstream kinases CHK1 and WEE1 to inhibit CDK1, thereby halting entry into mitosis. Our transcriptomic profiling revealed a marked induction of homologous recombination (HR)-associated double-strand break repair genes, including BRCA1, BRCA2 and RAD51, in p53-deficient cells following TAS102-PARPi treatment. This pronounced upregulation likely reflects enhanced DNA repair activity at the G2 checkpoint, orchestrated by ATR kinase. Based on this mechanistic, we tested whether the TAS102-PARPi regimen could be potentiated by targeting G2-checkpoint kinases. We developed a triple-drug therapeutic strategy that combines our two-drug regimen with a G2-checkpoint kinase inhibitor. Subsequent inhibition of checkpoint kinases such as WEE1 or ATR releases the G2-arrested cells into mitosis, resulting in mitotic catastrophe and cell death. Importantly, sequential administration, delaying the G2-kinase inhibitor after TAS102-PARPi, allows p53WT cells time to repair DNA, thereby minimizing toxicity to normal tissues. This sequential triple-drug strategy acts through a synthetic lethality mechanism, producing massive cell death in p53mut cancer models. In PDXs models, this regimen achieved robust tumor suppression without detectable toxicity. Citation Format: Mohammed M. Alruwaili, Yanqi Guo, Justin Zonneville, Thomas Melendy, Robert M. Straubinger, Barbara A. Foster, Priyanka Rajan, Henry G. Withers, Sarah Chatley, Renuka V. Iyer, Christos Fountzilas, Andrei V. Bakin. A novel therapeutic approach for targeting p53-mutant cancers by leveraging DNA damage response vulnerabilities [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 6762.

Magnetic chromatography was exploited to fractionate suspensions of magnetoliposomes (SML: lumen-free lipid-encapsulated clusters of multiple magnetic iron-oxide nanoparticles) improving their colloidal properties and relaxivity (magnetic resonance image contrast capability). Fractionation (i) removed sub-populations that do not contribute to the MRI response, and thus (ii) enabled evaluation of the size-dependence of relaxivity for the MRI-active part, which was surprisingly weak in the 55-90 nm range. MC was therefore implemented for processing multiple PEGylated SML types having average sizes ranging from 85 to 105 nm, which were then shown to have strongly size-dependent uptake in an in vivo pancreatic cancer model. Hence for applications in cancer diagnosis, selection of SML of suitable size for the biological target is more important than size-dependence of relaxivity.

The tumor suppressor TP53 gene (p53) is mutated in most human malignancies; however, existing treatment options are largely ineffective, lack selectivity, and cause toxic side effects. To address these clinical problems, we developed a sequential triple-drug strategy for p53 mutant cancer cells. Here we show that a combination of a thymidine analogue (TAS102) plus PARP inhibitor (PARPi) promotes formation of DNA double-strand breaks (DSBs) and G2-arrest specifically in p53 mutant cancer cells. Transcriptome analysis revealed that TAS102-PARPi treatment of p53 mutant cells did not repress DNA replication but activated DSB repair and blocked the mitotic program, consistent with G2-arrest. In contrast, TAS102-PARPi treatment of normal p53 wild-type cells resulted in a temporal G1-arrest and rapid recovery of cell cycle capacity after drug withdrawal. In p53 mutant cancer cells, subsequent blocking of a G2-checkpoint kinase, such as WEE1, released these G2-arrested cells into mitosis, leading to massive cell death. Delayed administration of a G2-kinase inhibitor provides time for p53 wild-type cells to repair DNA, thereby minimizing toxicity to normal tissues. This sequential triple-drug strategy exhibited robust efficacy in preclinical models of colorectal and pancreatic cancers and was well tolerated in mice. Together, our findings illustrate a promising triple-drug strategy for targeting p53 mutant malignancies.

Nanomedicines have seen widespread use in several malignancies including pancreatic cancer where standard-of-care chemotherapy regimens regularly employ nanomedicines including nab-paclitaxel and liposomal irinotecan. However, a small fraction of the administered dose reaches the tumor, and that fraction rarely perfuses beyond the tumor periphery due to the dense desmoplasia reaction from cancer-associated fibroblasts. Sonidegib, an inhibitor of the sonic hedgehog signaling axis, transiently deconstructs delivery barriers in pancreatic tumor xenografts by targeting myofibroblasts thus priming tumors for nanoparticle deposition. However, these effects are heterogeneous and often short-lived due to rebound fibrosis. We demonstrated that co-priming with plerixafor, an inhibitor of the C-X-C motif type-4 receptor, prevents priming resistance onset via several mechanisms including inhibition of hedgehog signaling crosstalk between tumor and stromal cells and reversal of epithelial-to-mesenchymal transition. Plerixafor was shown to block rebound hedgehog ligand production by downstream MEK-ERK signaling which interrupted hedgehog signaling feedback to cancer-associated fibroblasts. Immunofluorescence staining revealed decreased expression of classical mesenchymal markers in co-cultures of pancreatic cancer cells and fibroblasts treated with plerixafor after repeated exposure to sonidegib. Importantly, when administered concurrent with sonidegib, plerixafor increased deposition of fluorescently-labeled liposomes suggesting an enhancement of the tumor priming effect compared to sonidegib priming alone. We surmise that co-priming via sondegib and plerixafor may enhance exposure to nanoparticle-associated chemotherapeutics in highly desmoplastic pancreatic tumors compared to sonidegib priming alone. This combination may be explored further to probe anti-tumor efficacy when combined with nanoparticle-associated chemotherapy currently employed for treating pancreatic cancer. Future investigations will evaluate the pro-immune effects of plerixafor in this co-priming strategy to improve outcomes with contemporary immune checkpoint therapeutics. Jonathan R. Perri, Andrea Serratore, Ninfa L. Straubinger, Robert M. Straubinger. Co-priming sonic hedgehog inhibitor sonidegib with plerixafor induces tumor microenvironment transformation that enhances fluorescent nanoparticle deposition in pancreatic tumor xenograft [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 A117.

Pancreatic ductal adenocarcinoma (PDAC) is often chemotherapy-resistant, and novel drug combinations would fill an unmet clinical need. Previously we reported synergistic cytotoxic effects of gemcitabine and trabectedin on pancreatic cancer cells, but underlying protein-level interaction mechanisms remained unclear. We employed a reliable, sensitive, comprehensive, quantitative, high-throughput IonStar proteomic workflow to investigate the time course of gemcitabine and trabectedin effects, alone and combined, upon pancreatic cancer cells. MiaPaCa-2 cells were incubated with vehicle (controls), gemcitabine, trabectedin, and their combinations over 72 hours. Samples were collected at intervals and analyzed using the label-free IonStar liquid chromatography-mass spectrometry (LC-MS/MS) workflow to provide temporal quantification of protein expression for 4,829 proteins in four experimental groups. To characterize diverse signal transduction pathways, a comprehensive systems pharmacodynamic (SPD) model was developed. The analysis is presented in two parts. Here, Part I describes drug responses in cancer cell growth and migration pathways included in the full model: receptor tyrosine kinase- (RTK), integrin-, G-protein coupled receptor- (GPCR), and calcium-signaling pathways. The developed model revealed multiple underlying mechanisms of drug actions, provides insight into the basis of drug interaction synergism, and offers a scientific rationale for potential drug combination strategies.

Supplementary Table S5. Gene set enrichment analysis of genes differentially expressed in sHHi treated PDX #18269 based on GO annotations

Abstract Colorectal Cancer (CRC) is the third most common cause of cancer mortality in the US. A major problem in CRC management is relapse and progression to metastatic disease leading to poor overall survival. The tumor suppressor p53 is mutated in most CRCs and often drives metastasis and therapy resistance. Current therapeutic options targeting p53 mutant cancers show poor efficacy and frequently exhibit high toxicity. To address this clinical problem, we developed a novel therapeutic strategy for selective targeting of p53 mutant cancers. The strategy combines a thymidine analogue (e.g., trifluorthymidine/TFT, a component of TAS102) and poly (ADP-ribose) polymerase inhibitor (PARPi). We observed that TAS102 does not block DNA replication, but rather prompts post-replicative base-excision DNA repair (BER), and PARPi increases double-strand DNA breaks (DSBs) in p53 mutant cancer cells. Normal p53 wild type (p53wt) cells are arrested in the G1 phase and repair DNA. Thus, the TAS102-PARPi combination selectively targets p53-mutant cancer cells, resulting in the accumulation of DSBs and cell death. This novel strategy was examined in p53wt and p53-mutant tumor cell-derived xenograft (CDX) and patient-derived xenograft (PDX) models. The drug combination was significantly more effective in inhibiting p53 mutant tumor growth and in prolonging survival compared to monotherapies. This strategy is currently being tested in a first-in-human dose-escalation Phase I study (NCT04511039) in patients with advanced CRC. Further investigation revealed that the TAS102-PARPi combination induced DNA damage response (DDR) and the G2 checkpoint, leading to G2 arrest in p53 mutant cancer cells. We observed that TAS102-PARPi induced phosphorylation of CDC2 (also known as CDK1) but did not increase phosphorylation of histone H3 (a marker of mitosis), therefore indicating activation of the G2 checkpoint. Furthermore, the roles of ATM and ATR kinases in the DDR and G2 responses were clarified using highly specific kinase inhibitors. The analysis of the DDR/G2 signaling response in isogenic p53wt and mutant CRC cell lines showed that inhibition of WEE1 kinase can selectively abrogate the G2 checkpoint in p53-mutant cells, resulting in marked DNA damage and cell death. Importantly, the blockade of WEE1 strongly synergized with the TAS102-PARPi combination, further enhancing its potency and efficacy against p53-mutant cancer cells. In summary, our research provides a foundation for a novel therapeutic strategy for p53 mutant cancers that may provide a paradigm shift in the treatment of these deadly cancers, increasing efficacy while minimizing toxic adverse effects. Citation Format: Mohammed M. Alruwaili, Natsumi Naranjo, Priyanka Rajan, Kyeong Beom Jo, Dae-Kyum Kim, Thomas Melendy, Robert Straubinger, Christos Fountzilas, Andrei Bakin. Novel therapeutic approach for targeting p53 mutant colorectal cancers by affecting post-replicative DNA repair [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 7590.

Supplementary Figure S7. Kinase enrichment analysis of differentially-expressed genes after SHHi treatment

The tumor-suppressor p53 is commonly inactivated in colorectal cancer and pancreatic ductal adenocarcinoma, but existing treatment options for p53-mutant (p53Mut) cancer are largely ineffective. Here, we report a therapeutic strategy for p53Mut tumors based on abnormalities in the DNA repair response. Investigation of DNA repair upon challenge with thymidine analogs reveals a dysregulation in DNA repair response in p53Mut cells that leads to accumulation of DNA breaks. Thymidine analogs do not interrupt DNA synthesis but induce DNA repair that involves a p53-dependent checkpoint. Inhibitors of poly(ADP-ribose) polymerase (PARPis) markedly enhance DNA double-strand breaks and cell death induced by thymidine analogs in p53Mut cells, whereas p53 wild-type cells respond with p53-dependent inhibition of the cell cycle. Combinations of trifluorothymidine and PARPi agents demonstrate superior anti-neoplastic activity in p53Mut cancer models. These findings support a two-drug combination strategy to improve outcomes for patients with p53Mut cancer.

Supplementary Table S4. Gene set enrichment analysis of genes differentially expressed in sHHi treated PDX #18269 based on Hallmark genes in the MSig database

Supplementary Figure S4. Daily SHHi treatment drives EMT polarization

AimsPancreatic ductal adenocarcinoma (PDAC) is often intrinsically-resistant to standard-of-care chemotherapies such as gemcitabine. Acquired gemcitabine resistance (GemR) can arise from treatment of initially-sensitive tumors, and chemotherapy can increase tumor aggressiveness. We investigated the molecular mechanisms of chemoresistance and chemotherapy-driven tumor aggressiveness, which are understood incompletely.MethodsDifferential proteomic analysis was employed to investigate chemotherapy-driven chemoresistance drivers and responses of PDAC cells and patient-derived tumor xenografts (PDX) having different chemosensitivities. We also investigated the prognostic value of FGFR1 expression in the efficacy of selective pan-FGFR inhibitor (FGFRi)-gemcitabine combinations.ResultsQuantitative proteomic analysis of a highly-GemR cell line revealed fibroblast growth factor receptor 1 (FGFR1) as the highest-expressed receptor tyrosine kinase. FGFR1 knockdown or FGFRi co-treatment enhanced gemcitabine efficacy and decreased GemR marker expression, implicating FGFR1 in augmentation of GemR. FGFRi treatment reduced PDX tumor progression and prolonged survival significantly, even in highly-resistant tumors in which neither single-agent showed efficacy. Gemcitabine exacerbated aggressiveness of highly-GemR tumors, based upon proliferation and metastatic markers. Combining FGFRi with gemcitabine or gemcitabine+nab-paclitaxel reversed tumor aggressiveness and progression, and prolonged survival significantly. In multiple PDAC PDXs, FGFR1 expression correlated with intrinsic tumor gemcitabine sensitivity.ConclusionFGFR1 drives chemoresistance and tumor aggressiveness, which FGFRi can reverse.

Supplementary Figure S9. nuFGFR1 binds oncogenic pathway promoters that are also upregulated by SHHi

Supplementary Table S3. Genes differentially expressed by sHHi treatment in SHHi-responder PDX #18269

Supplementary Figure S3. SSL deposition in PDXs #18269, #14312, and non-responsive PDX #18254

Supplementary Figure S8. Correlation of FGFR1 expression with EMT marker expression in PDAC patient tumors

Background and PurposeElevated fibroblast growth factor receptor (FGFR) activity correlates with pancreatic adenocarcinoma (PDAC) progression and poor prognosis. However, its potential as a therapeutic target remains largely unexplored.Experimental ApproachThe mechanisms of action and therapeutic effects of selective pan-FGFR inhibitors (pan-FGFRi) were explored using in vitro and in vivo PDAC models ranging from gemcitabine-sensitive to highly gemcitabine-resistant (GemR). Gain-/loss-of-function investigations were employed to define the role of individual FGFRs in cell proliferation, migration, and treatment response and resistance.ResultsThe pan-FGFRi NVP-BGJ398 significantly inhibited cell proliferation, migration, and invasion, and downregulated key cell survival- and invasiveness markers in multiple PDAC cell lines. Gemcitabine is a standard-of-care for PDAC, but development of resistance to gemcitabine (GemR) compromises its efficacy. Acquired GemR was modelled experimentally by developing highly GemR cells using escalating gemcitabine exposure in vitro and in vivo. FGFRi treatment inhibited GemR cell proliferation, migration, GemR marker expression, and tumour progression. FGFR2 or FGFR3 loss-of-function by shRNA knockdown failed to decrease cell growth, whereas FGFR1 knockdown was lethal. FGFR1 overexpression promoted cell migration more than proliferation, and reduced FGFRi-mediated inhibition of proliferation and migration. Single-agent FGFRi suppressed the viability and growth of multiple patient-derived xenografts inversely with respect to FGFR1 expression, underscoring the influence of FGFR1-dependent tumour responses to FGFRi. Importantly, secondary data analysis showed that PDAC tumours expressed FGFR1 at lower levels than in normal pancreas tissue.Conclusions and ImplicationsSingle-agent FGFR inhibitors mediate selective, molecularly-targeted suppression of PDAC proliferation, and their effects are greatest in PDAC tumours expressing low-to-moderate levels of FGFR1. image

Therapeutic antibodies have shown little efficacy in the treatment of pancreatic ductal adenocarcinomas (PDAC). Tumor desmoplasia, hypovascularity, and poor perfusion result in insufficient tumor cell exposure, contributing to treatment failure. Smoothened inhibitors of hedgehog signaling (sHHi) increase PDAC tumor permeability, perfusion, and drug delivery, and provide a tool to develop a quantitative, mechanistic understanding as to how the temporal dynamics of tumor priming can impact intratumor distribution of monoclonal antibodies (mAb). A linked pharmacokinetic (PK)/pharmacodynamic (PD) model was developed to integrate the plasma and tumor PK of a sHHi priming agent with its effects upon downstream stromal biomarkers Gli1, hyaluronic acid, and interstitial fluid pressure in PDAC patient-derived xenograft (PDX) tumors. In parallel, in situ tumor concentrations of cetuximab (CTX: anti-epidermal growth factor receptor; EGFR) were quantified as a marker for tumor delivery of mAb or antibody-drug conjugates. A minimal, physiologically-based pharmacokinetic (mPBPK) model was constructed to link sHHi effects upon mechanistic effectors of tumor barrier compromise with the intratumor distribution of CTX, and CTX occupancy of EGFR in tumors. Integration of the mPBPK model of mAb deposition and intratumor distribution with the PK/PD model of tumor responses to priming not only identified physiological parameters that are critical for tumor antibody distribution, but also provides insight into dosing regimens that could achieve maximal tumor disposition of therapeutic antibodies under conditions of transient PDAC tumor permeability barrier compromise that mechanistically-diverse tumor priming strategies may achieve.(c) 2023 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.

Supplementary Figure S6. Analysis of transcription factor and master regulator enrichment of differentially expressed genes predict kinase-driven transcriptional changes in SHHi-treated tumors

IntroductionImmunogenicity continues to be a challenge for development and clinical utility of monoclonal antibodies, and there are gaps in our current ability to prevent anti-drug antibody development in a safe and antigen-specific manner.MethodsTo mitigate immunogenicity of monoclonal antibodies administered subcutaneously, O-phospho-L-serine (OPLS)—the head group of the tolerance-inducing phospholipid, phosphatidylserine—was investigated as an immunoregulatory adjuvant.ResultsFormulations of adalimumab, trastuzumab or rituximab with OPLS showed reduction in relative immunogenicity in mice compared to vehicle formulations, indicated by reduced anti-drug antibody development and significant reductions in CD138+ plasma cell differentiation in bone marrow. Titer development toward recombinant human hyaluronidase, a dispersion enhancer that was co-formulated with monoclonal antibodies, was similarly reduced. Subcutaneous administration of adalimumab with OPLS resulted in a two-fold increase in expression of type 1 regulatory (Tr1) T cell subset in the spleen. This is consistent with in vitro studies where co-culturing of dendritic cells primed with ovalbumin in the presence and absence of OPLS and antigen specific T-cells induced expression of Tr1 phenotype on live CD4+ T cells.ConclusionThis adjuvant does not impact immune competence of non-human primates and mice, and repeated administration of the adjuvant does not show renal or hepatic toxicity. Formulation of monoclonal antibodies with the immunoregulatory adjuvant, OPLS, was found to be safe and effective at mitigating immunogenicity.