Molecular Cancer Therapeutics

Pancreatic ductal adenocarcinoma (PDAC) is a devastating malignancy with a five-year survival rate of approximately 13%, underscoring the urgent need for novel therapeutic strategies. Next-generation imipridones ONC206 and ONC212 are potent anticancer agents that activate the mitochondrial ClpP protease and the integrated stress response. Lurbinectedin, an FDA-approved therapy for metastatic small cell lung cancer, inhibits transcription by binding the DNA minor groove and has demonstrated preclinical efficacy in PDAC models. Here, we show that ONC206 and ONC212 are highly cytotoxic against PDAC cell lines as monotherapies and in combination with lurbinectedin. Both ONC206 and ONC212 achieved sub-micromolar seventy-two-hour IC values in BxPC-3, PANC-1, and HPAF-II PDAC cells, with ONC212 exhibiting greater potency across all lines. Mechanistically, ONC206 and ONC212 induce apoptosis through ClpX depletion, ATF4 induction, and caspase-mediated PARP cleavage. Combination treatment of lurbinectedin with both imipridones produced robust synergy, with ONC212 generally exhibiting stronger synergy at lower concentrations and HSA synergy scores up to 29.5. Importantly, these combinations showed minimal toxicity in CCD 841 CoN non-malignant colon epithelial cells, indicating selective tumor cell killing. Western blot analysis revealed that synergy between lurbinectedin and ONC212 is associated with upregulation of DR5 and downregulation of Bcl-2 and ClpX. These findings provide mechanistic and preclinical support for combining lurbinectedin with next-generation imipridones as a therapeutic strategy in PDAC.

Therapeutic resistance remains a barrier to durable treatment of colorectal cancer (CRC), highlighting the need to identify vulnerabilities in resistant tumor cells. REV7 (MAD2L2) is a multifunctional genome-maintenance protein with established roles in REV3-dependent translesion synthesis (TLS) as well as mitotic progression, spindle organization, and chromosome segregation. Although perturbing REV7 dependent DNA damage tolerance has emerged as a strategy to overcome chemoresistance, whether pharmacologic targeting of REV7 exposes vulnerabilities to mitotic disruption remains poorly understood. Here, we investigated the REV7 targeting small molecule inhibitor C10, which disrupts the REV7 and REV3 interaction, and found that C10 sensitized HCT116 and HT29 CRC cells to the microtubule-disrupting agent colchicine, resulting in markedly reduced clonogenic survival. In contrast, C10 did not substantially enhance sensitivity to cisplatin. Genetic loss of REV7 similarly increased sensitivity to colchicine, independently supporting a REV7 dependent vulnerability to microtubule disruption. Combined C10 and colchicine treatment was associated with pronounced alterations in mitotic and post-mitotic nuclear morphology, including condensed/mitotic-like and multinucleated configurations, and increased micronucleus formation. In HT29 cells, combined treatment also increased the frequency of cells containing multiple gamma tubulin positive foci, consistent with disrupted centrosome-associated organization. Together, these findings support a model in which REV7 dependent functions contribute to cellular tolerance and recovery from microtubule mediated mitotic stress, whereas REV7 and REV3 disruption renders CRC cells vulnerable to mitotic perturbation. This study broadens the potential consequences of targeting REV7 beyond its canonical role in TLS and suggests that combining REV7 directed inhibition with mitotic perturbation warrants further investigation as a therapeutic strategy in CRC and potentially other cancers.

Small cell lung cancer (SCLC) is an aggressive thoracic malignancy with a 5-year survival rate under 7%. Lack of meaningful improvement of survival rates despite advances in treatment highlights the need for novel therapeutic approaches to improve patient outcomes. Currently, carboplatin + etoposide chemotherapy is the backbone of treatment for most patients. Lurbinectedin is a cytotoxic drug with unique activity against small cell lung cancers in patients with extensive disease and acquired resistance to carboplatin + etoposide. Our preliminary experiments in human SCLC cell lines treated with lurbinectedin demonstrated a dose-dependent increase in Chk1 and Chk2 protein phosphorylation. A consequence of the frequent TP53 inactivation in SCLC is tumor cell reliance on G2/M cell cycle checkpoints involving Chk1/Chk2 to maintain genomic integrity and allow cell survival following DNA damage. We hypothesised that inhibition of Chk1/Chk2-dependent responses with dual-inhibitor prexasertib (ACR-368), would potentiate tumor cell killing by lurbinectedin potentially in a synergistic manner. SCLC cells underwent cell death following single agent prexasertib exposure and this further increased with prexasertib + lurbinectedin combination. Highest Single Agent (HSA) synergy score calculations based on cell viability measurements suggested synergistic action between prexasertib and lurbinectedin at select dose combinations. Western blot analysis of intracellular proteins from SCLC cells treated with both drugs demonstrate dynamic, dose-dependent effects on Chk2, Chk1 and downstream effector Wee1, with lurbinectedin increasing intracellular levels of pChk1 and pChk2, while co-treatment with prexasertib deregulates this process across multiple human-derived cell lines. Synergistic killing was associated with elevated {gamma}-H2AX levels indicative of DNA double strand breaks and PARP-cleavage due to apoptotic caspase activation. Despite some heterogeneity among treated SCLC cells, the increased phosphorylation of Chk1 was noted at several kinase-activating sites including Serine 296, 317, and 345 while Chk2 Tyrosine 68 phosphorylation was consistently upregulated by lurbinectedin. The results provide a preclinical mechanistic rationale for overcoming a pro-survival, drug resistance- promoting checkpoint pathway to enhance the unique efficacy of single-agent lurbinectedin in patients with SCLC.

Resistance limits the clinical efficacy of RAS inhibitors. We applied chemical and genetic screens and identified the AXL receptor tyrosine kinase as a driver of resistance to RAS-ERK inhibition. We determined that combination treatment with the AXL inhibitor bemcentinib (AXLi) together with the RAS(ON) multi-selective tri-complex inhibitor RMC-7977 (RASi) or the ERK-selective inhibitor SCH772984 (ERKi) significantly enhanced growth suppression in human KRAS-mutant pancreatic and lung cancer models. Combined AXLi and RASi treatment of human KRAS-mutant pancreatic cell line-derived xenograft tumors synergistically suppressed ERK activation and MYC expression, and caused tumor regression. Analyses of immunocompetent mouse allograft pancreatic tumor models revealed a largely tumor cell-intrinsic response to inhibitor treatment. We identified an unexpected mechanism whereby KRAS inhibition upregulated the AXL ligand GAS6, activating AXL but inducing an AXL-dependent adaptive resistance mechanism wherein AXL antagonizes RASi efficacy. Our observations support concurrent AXL inhibition as a strategy to enhance RAS inhibitor clinical efficacy. STATEMENT OF SIGNIFICANCEOur findings identify AXL as a driver of resistance to RAS inhibitors, establishing a combination strategy to overcome resistance and enhance RAS inhibitor therapeutic efficacy in KRAS-mutant cancer by maximally inhibiting oncogenic RAS signaling.

Prostate cancer (PCa) is the most commonly diagnosed cancer and the second leading cause of cancer death among US men. Metastatic castration-resistant PCa (mCRPC) is a clinically advanced form of PCa, often associated with increased aggressiveness, metastatic potential, morbidity, and a higher risk of developing resistance to taxanes (TX), the first-line chemotherapy for mCRPC. Furthermore, cancer stemness and epithelial to mesenchymal trans-differentiation (EMT) potentially contribute to aggressiveness and the development of drug resistance in mCRPC. Here, we applied our drug development pipeline, secDrug, which utilizes a pharmacogenomics data-driven computational algorithm, to demonstrate that TAK-715 - a dual inhibitor of p38 MAPK and Casein kinase {delta}/{epsilon}, is a promising treatment for these advanced/lethal variants of PCa. Using in vitro cytotoxicity assays followed by cell-based functional assays in a panel of mCRPC cell lines representing TX-sensitive mCRPC, clonally derived TX-resistant lines, and a highly aggressive metastatic variant of mCRPC, we demonstrated the efficacy of TAK-715 treatment as a single agent and in combination with TX, including cancer stem-like cells. Further, we showed that the apoptotic effects of TAK-715 occur via a mitochondrial-mediated pathway. Bulk tumor RNA sequencing followed by pathway analysis identified genes associated with mitochondrial dysfunction and cell cycle arrest as the top molecular networks associated with TAK-715 single-treatment. HES1, a gene associated with nodal metastasis and PCa progression, was the top differentially expressed gene. Single-cell transcriptomics analysis revealed that TAK-715 treatment erodes the subclonal populations responsible for cancer stemness, metastasis, and drug resistance. The clinical significance of these findings was validated in silico using multiple patient datasets. Our results suggest that TAK-715 treatment has the potential to decrease oncogenic progression and cancer stem cell-like activity in drug-resistant, aggressive, and stem-like mCRPC cells.

Background: 177Lu-PSMA-617 radioligand treatment represents a major advancement in the management of metastatic castration-resistant prostate cancer (mCRPC), but key questions remain about tumor-cell dynamics and evolution in patients who have received this drug. Here, we used a circulating tumor cell (CTC) capture assay to assess the prognostic capacity of CTC subset dynamics following 177Lu-PSMA-617 treatment in a cohort of mCRPC patients. Methods: Using an AI-empowered holographic imaging platform combined with in-flow protein marker analysis, we prospectively analyzed serial CTC samples from patients receiving 177Lu-PSMA-617 therapy at pre-treatment and on-treatment time points (n=39 of 100 enrolled patients). PSMA and EpCAM proteins in CTCs were determined by immunofluorescence staining. Proportions and absolute counts of CTC subsets delineated by PSMA and EpCAM proteins were assessed in PSA50 responders (n=19) and non-responders (n=20). Within-patient changes in CTC subsets were compared using Wilcoxon signed-rank tests with Benjamini-Hochberg correction. Associations of CTC subset abundance and dynamics with overall survival were assessed using Kaplan-Meier estimates with log-rank tests and univariable Cox proportional hazards models. Results: PSMA and EpCAM stratified CTCs into subsets, some of which were associated with survival outcomes following 177Lu-PSMA-617. We observed an increase in the proportion of PSMA-/EpCAM+ CTCs following 177Lu-PSMA-617, with a larger increase occurring in PSA50 responders (6.2% to 18.7%; p=0.041) than non-responders (7.9% to 15.4%; p=0.023). This corresponded to a decrease in the proportion of PSMA+/EpCAM- CTCs, whereas the proportion of PSMA+/EpCAM+ CTCs did not decline. On-treatment counts of >5 CTCs, relative to [≤]5 CTCs, were associated with shorter overall survival in the PSMA+/EpCAM+ (HR 3.56, 95%CI 1.32-9.60, p=0.012) and PSMA+/EpCAM- (HR 7.50, 95%CI 0.96-52.99, p=0.054) subsets. Finally, on-treatment declines in PSMA+/EpCAM+ (HR 0.74, p=0.49) and PSMA+/EpCAM- (HR 0.61, p=0.215) CTC subsets were not significantly associated with overall survival relative to sustained high levels of these cells. Conclusions: In mCRPC patients receiving 177Lu-PSMA-617, dynamic changes in CTC subsets delineated by PSMA and EpCAM expression may have prognostic value, justifying additional evaluation.

Testicular germ cell tumors (TGCTs) are highly curable malignancies; however, resistance to cisplatin-based therapy remains a critical clinical challenge. G-quadruplexes (G4s) are specific guanine rich, four-stranded DNA structures that are enriched at distinct genomic regions. Due to their stability, G4s can influence genome stability, gene expression, DNA replication as well as telomere maintenance. G4 stabilization by small molecules has been shown to have anticancer effects in several tumors but remains underexplored in TGCTs. Here, we systematically evaluated the effect of six different G4- ligands (CX-5461, CX-3543, BRACO19, PDS, CM03 and RHPS4) on TGCT cell lines and non-malignant fibroblasts. Among those G4 ligands, CX-5461 showed strongest cellular changes in TGCTs. We observed dose- and time-dependent G4 stabilization upon CX-5461 treatment in TGCT cells, accompanied by impaired RNA polymerase I-dependent transcription and induction of DNA double-strand breaks, whereas only minimal effects were observed in control fibroblasts. These effects led to the activation of p53 signaling, and consequently an upregulation of canonical p53 target genes, including CDKN1A, MDM2, PIDD1, GADD45A, ZMAT3, SESN1 and PPM1D, resulting in G2/M cell-cycle arrest and apoptosis. The presence of potential quadruplex forming sequences (PQSs) in the promoters or gene bodies of these genes suggests that G4 structures may contribute to the p53 transcriptional response. Collectively, these data demonstrate that CX-5461 appears as highly potent G4-ligand inducing p53-dependent molecular cascades leading to cell death in germ cell tumors. Unlike in many other tumors, p53 remains functionally intact in TGCTs, suggesting that CX-5461-mediated activation of the p53 pathway could be selectively targeted as a therapeutic strategy in TGCTs.

In this study, we identify Topoisomerase II (TopII ) targeting and MUS81 deficiency as sensitisers to trastuzumab deruxtecan (T-DXd), a HER2-targeting antibody-drug conjugate with a potent topoisomerase I poison payload that is of clinical importance in the treatment of HER2 expressing solid tumours in a range of cancer types. Using preclinical tumour spheroid models of breast and colorectal cancer, we show that TopII targeting with doxorubicin sensitises HER2-low tumour cells to T-DXd, elevating cell cytotoxicity, DNA damage, and checkpoint pathway activation. T-DXd treatment, both as a single agent and in combination with doxorubicin, increases TopII expression, highlighting this nuclear endonuclease as a potential candidate biomarker for T-DXd response in both the HER2-high and HER2-low setting. Using isogenic CRISPR models, we show that genetic loss of the MUS81 structure-specific endonuclease, a key processor of branched DNA structures and under-replicated DNA, sensitises HER2-low colorectal cancer cells to T-DXd. Given that reduced expression of MUS81 is closely related to metastasis and poor prognosis in colorectal carcinoma, our findings highlight the potential utility of these treatment combinations in a subset of colorectal cancer patients that present with HER2-positve/MUS81-low disease.

Radiotherapys clinical utility is often limited by radio-resistance, enterotoxicity, and intestinal dysbiosis. This study evaluated pyrogallol--a plant-derived vicinal trihydroxybenzene--as a dual-action radiosensitizer and mucosal protectant in an Ehrlich ascites carcinoma (EAC) BALB/c mouse model subjected to targeted LINAC irradiation (8 Gy). By combining transcriptomic profiling with whole-genome metagenomic sequencing, we interrogated the underlying host-microbiome interactions. Pyrogallol co-treatment significantly augmented radiotherapeutic efficacy, driving marked tumour regression through the upregulation of pro-apoptotic effectors (Bax, Casp3, Casp7) and p53-mediated tumour suppressors (Tp53, p21), alongside Bcl2 repression. Concurrently, pyrogallol blunted oncogenic progression by arresting proliferation (Cdk4, Pcna), inhibiting epithelial-mesenchymal transition (N-cadherin, vimentin), downregulating fibrotic remodelling (Tgf-{beta}, Col1A1, Fibronectin), and attenuating radiation-induced pro-inflammatory cytokine surges (Il-1, Il-6, Il-12). At the gut interface, radiation degraded colonization resistance by depleting homeostatic short-chain fatty acid producers and Clostridium scindens, while fuelling pathobiont blooms (Acinetobacter baumannii, Clostridioides difficile). Pyrogallol reversed this dysbiosis through a distinct ecological shift; despite a reduction in total species richness, the intestinal niche became dominated by the next-generation probiotic Parabacteroides distasonis ([~]94% relative abundance; Berger-Parker index: 0.94). Integrated Spearmans rank correlations demonstrated that host proliferative, EMT, fibrotic, and inflammatory markers aligned positively with pathobiont clusters (Bacteroides caecimuris, B. faecium, A. baumannii). Conversely, tumour regression and anti-inflammatory signatures correlated strongly with pathobiont restriction and P. distasonis enrichment. Overall, pyrogallol emerges as a compelling therapeutic adjuvant that synergistically enhances tumour radiosensitivity while remodelling the gut microbiome into a protective, anti-inflammatory state.

The Hippo pathway is an evolutionarily conserved regulator of growth, regeneration, and organ homeostasis, and while its dysregulation is well established in cancer, the effects of inhibiting this pathway on normal tissues are less understood. Here we have systematically investigated the impact of Hippo pathway inhibition by comparing pharmacologic perturbation using a covalent small-molecule TEAD inhibitor (TEADi CMPD1, also known as GNE-8025) with genetic suppression of YAP/TAZ. We identified three key target organs that consistently emerged upon TEAD inhibition: the kidney, as well as the pancreas, and thymus. Across models, both perturbations led to comparable disease phenotypes in these organs, including tubular degeneration in the kidney, acinar atrophy in the pancreas, and lymphoid depletion in the thymus. However, the extent of damage was more pronounced in mice treated with the small-molecule inhibitor, highlighting potential dose and compound specific effects while remaining broadly consistent with the phenotypes observed upon genetic ablation of YAP/TAZ. This highlights the key role of evaluating both genetic and pharmacological perturbations to characterize the phenotypes and potential toxicities when modulating novel targets in oncology. To further investigate the mechanisms underlying pan-TEAD inhibition and kidney related adverse effects, we further characterized this class effect through a comprehensive transcriptomic analysis of the kidney to map the pathways involved in renal response. SignificanceUnderstanding on target toxicities is critical for the safe clinical development of TEAD inhibitors. Here, by integrating pharmacologic TEAD inhibition with genetic suppression by developing a mouse model that characterizes systemic, inducible knockdown of YAP/TAZ, we provide a systematic framework to define the Hippo pathway liabilities in vivo. We identify kidney, pancreas, and thymus as conserved target organs with concomitant phenotypes across both genetic and pharmacological methods, establishing these as pathway driven effects. Importantly, we uncover dose dependent and partially irreversible injury, particularly in kidney and pancreas, alongside mechanistic insight linking TEAD inhibition to aldosterone signaling disruption in kidney. These findings highlight the importance of strategies to identify monitorable, manageable adverse effect to guide clinical translation of TEAD targeting strategies.

Cisplatin remains as standard chemotherapy for patients with HNSCC, but rapid development of drug resistance has limited patient benefit. The p53 tumor suppressor plays a central role in the cellular response to DNA damage in cancer, triggering apoptosis to prevent propagation of damaged cells in tumor development. TP53 gene mutations occur in 65-86% of HNSCC. Small molecule PG3 induces the integrated stress response (ISR), leading to apoptosis via the HRI-eIF2-ATF4-PUMA axis. We hypothesized that a combination of PG3 plus cisplatin could increase apoptosis in TP53-mutated HNSCC cells through enhanced induction of the ISR and ATF4. PG3 synergized with cisplatin to inhibit cell viability, leading to potent apoptosis in TP53-deficient cells. The effect was regulated through the HRI-ATF4-NOXA pathway. Furthermore, we identified that cisplatin activates HRI and leads to the degradation of CReP (constitutive repressor of eIF2 phosphorylation) via E3 ligase {beta}-TrCP, contributing to the induction of the ISR. We noted decreased ATF4 levels after treatment with cisplatin, CPT, or PG3 and cisplatin. Thus, combined therapy of PG3 plus cisplatin likely results in adaptation and acquired resistance via degradation of ATF4. We targeted the degradation mechanism of ATF4 by inhibiting {beta}-TrCP1, CK1{delta}, or CK2, respectively. Each approach successfully blocked ATF4 degradation induced by cisplatin or PG3 plus cisplatin and enhanced apoptosis. Our results provide a rational strategy for triple treatments, involving an ISR inducer, a DNA damaging drug, and a {beta}-TrCP inhibitor/CK1{delta} inhibitor/CK2 inhibitor, to achieve potent and prolonged anti-tumor effects and overcome chemoresistance in HNSCC.

AbstractAdrenocortical carcinoma (ACC) is a rare and highly aggressive endocrine malignancy originating from the adrenal cortex with limited effective treatment options. The underlying pathophysiology of ACC is uniquely characterized by abnormal steroid production and increased metabolic activity, highlighting the critical role of mitochondria in adrenal steroid hormone biosynthesis and tumor metabolism. In this study, we investigated the therapeutic potential of TR-107, a novel and highly selective small-molecule agonist targeting the mitochondrial protease ClpP. Pharmacologic hyperactivation of ClpP disrupts mitochondrial proteostasis and bioenergetics and has shown promising antitumor activity in various preclinical models. Our results demonstrated that TR-107 induces potent dose-dependent cytotoxic effects at nanomolar concentrations in ACC cell lines NCI-H295R and mACC3 as well as short-term ACC patient-derived organoid (PDO) models, markedly reducing cell viability and confluency in vitro. Metabolic analyses revealed that TR-107 significantly impaired oxygen consumption, indicating a disruption of oxidative phosphorylation and substantial attenuation of basal cellular respiration. Mechanistic studies showed dose-dependent increases in reactive oxygen species (ROS) levels and upregulation of proteins involved in mediating the ferroptotic rheostat. Pharmacokinetic assessment uncovered that TR-107 was not a substrate of the ABCB1 (MDR1/P-glycoprotein) efflux transporter, suggesting potential to overcome common multidrug resistance mechanisms. Given the importance of IGF-2 signaling in ACC, we further explored the combinatorial effects of TR-107 with IGF-1 receptor (IGF-1R) inhibitors and discovered that co-treatment produced synergistic reductions in cell viability across NCI-H295R, mACC3, and ACC PDOs. Collectively, these findings support the potential of mitochondrial ClpP hyperactivation as a promising therapeutic strategy for ACC and demonstrate that TR-107 exhibits significant antitumor activity as a monotherapy or in combination with IGF-1R inhibitors. These findings provide a strong rationale for advancing ClpP agonists into clinical development for the management of ACC.

Background: Relapsed and refractory osteosarcoma (OS) remains a major therapeutic challenge, with fewer than 20% of patients surviving beyond 3 years. Increasing evidence indicates that MYC amplification/overexpression is associated with inferior survival. Small molecule inhibitors and immunotherapies have limited single-agent efficacy in pediatric solid tumors. Using syngeneic cell lines derived from p53-driven and MYC-hyperactivated genetically engineered mouse models (GEMMs) of OS, we tested cabozantinib, a multi-tyrosine kinase inhibitor with immunomodulatory properties, with TIGIT immune checkpoint blockade and investigated mechanisms underlying therapeutic response and resistance. Methods: In vitro cabozantinib sensitivity was established in GEMM-derived cell lines. Mice bearing tibial tumors were randomized to vehicle control, cabozantinib, anti-TIGIT antibody, or combination therapy, and tumor growth and survival assessed after a 3-week treatment period. Temporal RNA sequencing was performed at early (8-15 days) and late (18-24 days) time points to characterize transcriptomic changes associated with efficacy. Results: MYC-hyperactivated cell lines were more resistant to cabozantinib in vitro than p53-driven lines (mean IC50 5.51 vs 0.65 mciroM, p=0.0016). In p53-driven orthotopic models, combination therapy significantly decreased tumor growth and improved survival compared to solvent and cabozantinib alone, while in MYC-hyperactivated models cabozantinib-containing regimens delayed tumor progression relative to control or anti-TIGIT monotherapy, however the addition of anti-TIGIT did not significantly improve survival over cabozantinib alone. Temporal transcriptomics revealed upregulated anti-tumor immune-response pathways and decreased M2 macrophages only with combination treatment in the p53-driven model. In contrast, combination-treated MYC-hyperactivated models demonstrated increased TNF signaling and elevated Cxcl5 and Ccr2 expression, indicative of increased myeloid cell recruitment, and upregulation of extracellular matrix (ECM) remodeling pathways suggest a therapy-induced stress adapted state that propagates treatment resistance over time. Conclusion: New therapies are needed for patients with relapse or refractory OS. By targeting tumor-intrinsic resistance mechanisms and modulating the tumor microenvironment using cabozantinib and anti-TIGIT therapy, improved tumor control and survival was achieved in p53-driven orthotopic OS models. MYC-hyperactivated models were able to overcome therapeutic pressure and employ myeloid recruitment and ECM remodeling programs to achieve treatment resistance. Targeting of these programs should be considered in future studies investigating therapeutic strategies in relapsed and refractory OS.

Background: Trophoblast cell surface antigen 2 (TROP2, encoded by TACSTD2) is a transmembrane glycoprotein overexpressed in multiple aggressive epithelial carcinomas. While antibody drug conjugates targeting TROP2 have achieved regulatory approvals, acquired payload resistance and systemic off-target toxicities limit sustained remissions. Radionuclide Drug Conjugates (RDCs) represent a potent alternative modality capable of delivering cytotoxic ionizing radiation directly to target cells. However, selecting the optimal therapeutic radioisotope between long-range beta emitters (177Lu) and short-range, high linear energy transfer (LET) alpha emitters (225Ac) under heterogeneous TROP2 spatial distributions remains an unaddressed clinical challenge. Methods: We developed an automated computational pathology and spatial microdosimetry pipeline to resolve microscopic TROP2 expression gradients and simulate absorbed radiation dose distributions from digitized whole-tissue immunohistochemistry (IHC) sections (N = 14). Optical density matrices were de-convoluted in Hematoxylin-Eosin-DAB (HED) color space to isolate the DAB chromogen. Continuous 2D spatial density distributions and topological surface profiles were reconstructed. Physical radiation energy deposition was modeled using radial dose point kernels for 177Lu (mean range ~670 m, LET 0.2 keV/m) and 225Ac (mean range ~65 m, LET 100 keV/m, 4 alpha particles per decay cascade). Therapeutic Index (TI, ratio of mean target to non-target absorbed dose), target coverage, and spatial specificity were quantified across all specimens. Results: Quantitative image deconvolution revealed that TROP2 expression across the cohort was characteristically focal and clustered, with a mean positive area fraction of 1.55 +/- 2.22% (range: 0.08% to 6.85%) and mean DAB signal intensity of 0.256 +/- 0.043. In all 14 evaluated specimens (100%), 225Ac-labeled RDCs demonstrated superior tumor-to-stroma dose localization compared to 177Lu-labeled RDCs. The cohort-wide mean Therapeutic Index was significantly higher for 225Ac (1.26 +/- 0.14) than for 177Lu (1.01 +/- 0.02, p < 0.0001, paired two-tailed t-test). Because the path length of 177Lu beta particles exceeded target cell nest dimensions by up to 30-fold, 177Lu suffered from severe off-target crossfire spillover into antigen-negative stroma. In contrast, 225Ac confined high-LET ionization tracks strictly within the micro-geographic boundaries of TROP2-expressing clusters. Conclusions: In tumors displaying focal or sparse TROP2 micro-architecture, Targeted Alpha Therapy with 225Ac-RDCs offers a superior biophysical profile over beta-emitting 177Lu-RDCs, maximizing cluster cell kill while sparing adjacent normal tissue stroma. This computational microdosimetry framework provides a practical tool to guide rational isotope pairing in RDC drug design.

Background: Brain-penetrant histone deacetylase inhibitors (HDACi) are promising radiosensitizing agents for glioblastoma (GBM). HDAC inhibitors can impair DNA damage response (DDR) pathways and promote persistent DNA damage. Here, we evaluated whether combined inhibition of HDAC and the DNA repair kinase, ataxia-telangiectasia mutated (ATM), could synergize with radiation (IR) and improve therapeutic efficacy in GBM. Methods: In vitro dose-response assays were performed in patient-derived xenograft (PDX)-derived glioma stem cells (GSCs) with both inhibitors of HDAC (quisinostat, QST) and ATM (AZD1390) with or without IR treatment to assess synergy. Pharmacodynamic responses were evaluated by immunoblotting for histone acetylation, DDR signaling, and DNA damage markers. Transcriptomic analyses were performed to evaluate treatment-associated changes in DNA repair pathways. The combination of QST, AZD1390, and IR was further evaluated in an orthotopic GBM survival study. Results: Combined treatment with QST and AZD1390 produced synergistic radiosensitization in multiple GSC models in vitro. Furthermore, QST combined with IR suppressed transcription of homologous recombination (HR) pathway genes in GSCs, impairing double-strand break repair capacity through an epigenetic mechanism that does not directly inhibit ATM kinase activity. Pharmacodynamic analyses confirmed that QST increased histone acetylation while AZD1390 suppressed ATM signaling, together impairing complementary mechanisms to increase radiosensitivity of GSCs. In an orthotopic GBM model, QST combined with IR significantly prolonged survival compared with IR alone. However, the addition of AZD1390 did not provide a significant survival benefit beyond QST+IR, and AZD1390 combined with IR did not significantly improve survival relative to IR alone. Conclusions: Combined HDAC and ATM inhibition with QST and AZD1390 enhances radiosensitivity in patient-derived GBM cells, mechanistically underpinned by QST-mediated increase in DNA damage and dual disruption of DDR through complementary, non-overlapping mechanisms. However, this in vitro synergy did not confer additional survival benefit beyond QST+IR in an orthotopic GBM model under the dosing conditions tested, suggesting pharmacological limitations with the tumor microenvironment or suboptimal dosing schedules may have constrained in vivo efficacy.

Despite intensive treatment, high-risk neuroblastoma (HRNB) remains a leading cause of cancer-related mortality in children. Current treatment paradigms include multimodal systemic treatments and multi-agent chemotherapy, which is limited by substantial acute and long-term toxicities. Platinum-based chemotherapy is a cornerstone of this treatment but is fraught with side effects and stands to benefit from dose reduction strategies. MicroRNA (miR)-based therapeutics represent an attractive strategy to simultaneously moderate multiple oncogenic pathways, providing a potential avenue to enhance the efficacy of current treatment regimens. However, the development of miR therapeutics has largely relied on chemically synthesized ones, which are limited by issues of inconsistency and poor in vivo stability. Biologically produced, tRNA/pre-miR containing bioengineered miRs (bioRNAs) address these limitations. We investigated the therapeutic potential of bioRNA encoding miR-124 and miR-34a across molecularly distinct HRNB models and evaluated their ability to enhance cisplatin efficacy. Both bioRNAs significantly suppressed neuroblastoma cell viability in vitro, with proteomic analyses demonstrating preferential induction of neuronal differentiation by miR-124 and apoptotic signaling by miR-34a. Combining bioRNA with cisplatin produced robust synergy across multiple HRNB lines, enhancing apoptosis while simultaneously promoting differentiation-associated phenotypes. In vivo, both bioRNAs suppressed tumor growth as monotherapies, while combination with low-dose cisplatin produced the strongest responses across multiple xenograft models with different HRNB subtypes. Notably, low-dose cisplatin combined with bioRNA therapy frequently achieved tumor control comparable to, or greater than, that observed with high-dose cisplatin monotherapy while maintaining favorable tolerability. These findings establish bioRNAs as potent cisplatin sensitizers and support bioRNA-based combination therapy as a strategy for improving HRNB treatment while reducing chemotherapy-associated toxicity in children.

Purpose: There is an unmet need in the treatment of triple-negative breast cancer (TNBC), as recurrence rates remain high; thus, novel treatment strategies are warranted to improve outcomes for patients. Smac mimetics (SMs) are small molecules that inhibit the inhibitors of apoptosis. In this study, we investigated the properties of the SM Xevinapant (AT406) as a radiosensitizer in TNBC. Methods: TNBC cell lines were treated with Xevinapant (AT406) in the presence or absence of ionizing RT and cell viability and apoptosis measured in vitro. Clonogenic assays were performed to confirm radiosensitization. We also assessed xevinapant radiosensitization in the presence of the Tumor Necrosis Factor (TNF) cytokine, which has been shown to enhance SM-induced apoptosis. Radiosensitization mechanisms were further validated by western blotting, flow cytometry and cell cycle analysis. Results: We show that Xevinapant increased cell death in a panel of irradiated TNBC cell lines. In clonogenic assays, Xevinapant sensitized MDA-MB-231 cells to RT-induced cell death, and this was significantly enhanced in the presence of TNF, with a dose-enhancing factor (DEF) of 1.57. We also show that Xevinapant radiosensitization is mediated by TNF-dependent apoptosis. Conclusions: Xevinapant is a radiosensitizer that enhances RT-induced cell death in TNBC cells, and TNF is central to this process through activation of apoptosis. These results are promising for combining RT with Xevinapant to improve local control in TNBC.

Advanced-stage high grade serous ovarian cancer (HGSC) initially responds to platinum-based chemotherapy; however, most HGSC tumors recur and develop chemotherapy-resistance, making recurrent ovarian cancer (OC) a fatal disease. Ovarian cancer stem cells (OCSCs), a subpopulation of cells capable of surviving treatment and repopulating tumors, contribute to disease relapse, and understanding the mechanisms(s) responsible for OCSC survival is critical for developing strategies to overcome acquired resistance and prevent tumor recurrence. However, whether platinum-induced OCSC enrichment results from the selection of pre-existing OCSCs or chemotherapy-induced cellular plasticity, and the molecular mechanisms underlying these processes, remain poorly understood. Here, we demonstrated that cisplatin treatment not only enriched for OCSCs but promoted the conversion of ALDH- cells to ALDH+ cells, revealing chemotherapy-induced plasticity as a potential mechanism for OCSC expansion. This conversion was associated with nuclear receptor signaling pathways, vitamin D receptor signaling (VDR/RXR) and retinoic acid receptor signaling (RAR/RXR). Importantly, activation of VDR/RXR signaling with vitamin D reduced stemness phenotypes and the cisplatin induced conversion to ALDH+ cells. In addition, vitamin D combined with cisplatin decreased tumor volume in vivo compared to either treatment alone. In contrast, cisplatin treatment increased RAR/RXR signaling, supporting an associative role for ligand-dependent nuclear receptor signaling in OCSC plasticity. Together, these findings reveal that platinum chemotherapy can actively promote the acquisition of stem-like properties and identifies VDR/RXR signaling as a potential regulatory mechanism of OCSC plasticity. Targeting this pathway with vitamin D may provide a therapeutic strategy to target OCSCs and improve responses to platinum-based chemotherapy.

Background: Transcriptomic FOLFIRINOX-component sensitivity signatures have been clinically evaluated in resected and advanced PDAC, but their relevance and longitudinal stability in the neoadjuvant BR/LA setting are unknown. Patients and methods: We retrospectively studied 77 patients with borderline resectable or locally advanced PDAC treated with neoadjuvant FOLFIRINOX followed by resection. Pretreatment sensitivity to 5-fluorouracil, oxaliplatin and irinotecan was determined using previously developed locked transcriptomic classifiers and integrated into a regimen-level FOLFIRINOX classification. The primary molecular cohort comprised 53 patients whose pretreatment biopsies had [&ge;]10% tumour cellularity. Cox models assessed associations with survival. Longitudinal changes were assessed in 35 paired tumours. Results: Thirty-one pretreatment tumours were classified as FOLFIRINOX-sensitive (FFX-Sens), and 22 were not classified as FOLFIRINOX-sensitive (FFX-Res). FFX-Sens status was associated with longer overall survival (OS; hazard ratio [HR] 0.48, 95% confidence interval [CI] 0.25-0.93; p=0.029). In the complete-case model adjusted for baseline carbohydrate antigen 19-9 (CA19-9) and tumour size (n=50), the association with OS persisted (adjusted HR 0.47, 95% CI 0.23-0.96; p=0.037), whereas the adjusted association with disease-free survival was not statistically significant (adjusted HR 0.55, 95% CI 0.28-1.10; p=0.090). Among paired tumours, 15 changed from FFX-Sens to FFX-Res and four in the opposite direction (exact McNemar p=0.019). The OS association also persisted after adjustment for postoperative pathological factors (adjusted HR 0.40; p=0.015). Conclusions: Pretreatment FOLFIRINOX sensitivity was associated with OS under neoadjuvant FOLFIRINOX, while matched pretreatment and residual-tumour analyses revealed significant directional reclassification after treatment. These findings extend the clinical evaluation of validated drug-specific FOLFIRINOX sensitivity classifiers to the neoadjuvant setting and provide a longitudinal assessment of their stability during treatment. They support prospective evaluation of both pretreatment stratification and molecular reassessment of residual disease.

Radiotherapy induces cytotoxic DNA damage, but activation of DNA repair pathways and cell-cycle checkpoints can limit therapeutic efficacy. Here, we developed a high-throughput, flow cytometry-based screening platform to identify compounds that inhibit radiation-induced DNA repair and checkpoint activation. Reh leukemia and A549 lung cancer cells were irradiated and screened against up to 700 bioactive compounds, with DNA damage persistence quantified by {gamma}H2AX levels across independent screens. Cell barcoding using Pacific Blue staining was incorporated to enable highly accurate quantification of {gamma}H2AX across treatment conditions. The platform yielded robust and reproducible results and supported multiparametric analysis, including assessment of G2 checkpoint activation by phospho-histone H3. Largely overlapping candidate radiosensitizers were identified in both cell lines, including the multi-kinase inhibitor 5-iodotubercidin and the PI3K/mTOR inhibitor omipalisib. Validation studies in lung cancer and glioblastoma models confirmed screen performance. Mechanistically, omipalisib reduced phosphorylation of the non-homologous end-joining protein DNA-PK, consistent with impaired double-strand break repair. Both compounds enhanced radiosensitivity in clonogenic survival assays. Notably, 5-iodotubercidin increased radiosensitivity in glioblastoma cells despite previous reports of radioprotective effects in normal brain tissue. Together, these findings establish a robust barcoded screening approach for identifying radiosensitizers that target DNA damage repair and checkpoint responses.