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.

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.

Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) is a marker of cancer stem-like cells frequently upregulated in colorectal cancer (CRC) with lower expression in normal tissues, making it a favorable target for antibody-drug conjugates (ADCs). ADCs combine antibody specificity with potent cytotoxic payloads to enhance efficacy while minimizing systemic toxicity. LGR5-targeting ADCs incorporating different payloads demonstrate strong initial tumor inhibition, yet tumors eventually recur due in part to LGR5 downregulation, necessitating more effective strategies to prevent relapse. We show treatment with chemotherapies or an LGR5-targeting ADC coupled to a topoisomerase 1 inhibitor payload (8E11-CPT2) reduces LGR5 levels and increases MET receptor expression and/or activation in CRC cells, supporting a therapeutic approach to target LGR5 and MET simultaneously. Accordingly, we engineered a MET-targeting ADC (ABT-700-SG3199) via site-specific conjugation of the anti-MET antibody telisotuzumab (ABT-700) with the DNA-crosslinking pyrrolobenzodiazepine (PBD) dimer SG3199. ABT-700-SG3199 exhibited superior potency and efficacy in CRC models compared to the clinical-stage MET-targeting ADCs ABBV-399 and ABBV-400, which use the same antibody backbone conjugated to different payloads. Treatment with ABT-700-SG3199 increased LGR5 expression, and the combination of ABT-700-SG3199 with 8E11-CPT2 enhanced CRC cell-killing efficacy, reinforcing the rationale for a dual-targeting approach. In patient-derived xenografts, combined administration of 8E11-CPT2 and ABT-700-SG3199 markedly delayed tumor relapse and prolonged survival compared with single-agent treatment. Taken together, these findings reveal a reciprocal regulation between MET and LGR5 in response to LGR5- or MET-targeting ADCs in CRC models and support a LGR5/MET dual-targeting therapeutic strategy to enhance efficacy and potentially overcome resistance and relapse.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most challenging cancers to treat due to the dismal survival rate, poor post-treatment outcome and profound resistance to a wide range of therapies. With mutant KRAS being a key driver, small molecule inhibitors targeting KRAS or pan-RAS (KRASi) have demonstrated exciting preclinical and early clinical anti-tumor efficacy, and the pan-RAS(ON) inhibitor daraxonrasib (RMC-6236) recently achieved Phase 3 clinically meaningful improvements in patient survival compared to chemotherapy. But resistance to RAS/KRAS inhibitor inevitably develops, which limits and compromises the treatment outcome. In this study, we investigated the combination of stereotactic body radiation therapy (SBRT) and KRAS inhibition (MRTX1133 and daraxonrasib) in the treatment of preclinical PDAC models. We found that this combination strategy synergistically suppresses PDAC cell growth in vitro and enhances tumor control while minimizing local recurrence in orthotopically implanted KPC (LSL-KrasG12D/+;Trp53R172H/+;Pdx1-Cre) murine PDAC tumors in vivo. As radiation therapy (RT) induces ferroptosis in multiple cancer types and mutant KRAS promotes various anti-ferroptotic mechanisms, we tested the role of ferroptosis in promoting tumor-control efficacy. Intriguingly, the addition of a ferroptosis inhibitor, liproxstatin-1, to the combination therapy significantly abrogated the in vivo synergism between SBRT and KRAS inhibition, suggesting that treatment-induced ferroptosis at least partially drives the synergistic efficacy of this combination strategy. Our study indicates that this SBRT-KRASi combination has the potential to overcome treatment resistance and improve outcomes in PDAC patients. These data directly support the design of a planned multi-center Phase 2 clinical trial with this combination strategy in locally advanced PDAC.

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.

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: 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.

PurposeCisplatin (CDDP) is the most active chemotherapy for ovarian cancer; primary or acquired resistance signals a poor prognosis. Nelfinavir (NFV), an HIV protease inhibitor, has demonstrated anti-tumor activity in multiple cancer models, but its interaction with CDDP in ovarian cancer has yet to be demonstrated. In this work, we addressed whether the combination of CDDP and NFV provides treatment advantage in platinum (Pt)-resistant ovarian cancer cells. MethodsDrug synergy between NFV and CDDP was assessed using cell vitality assays and Loewe additivity modelling. Apoptotic and pyroptotic signalling were evaluated by immunoblotting, mitochondrial membrane potential analysis, and lactate dehydrogenase (LDH) release, and caspase inhibition. Transcriptomic changes were assessed by bulk mRNA sequencing followed by differential gene expression analysis and gene set enrichment analysis. ResultsNFV synergized with CDDP to reduce the viability of Pt-resistant ovarian cancer cells, promoting a regulated lytic cell death phenotype involving apoptotic and pyroptotic features. Combination treatment induced caspase-8 and caspase-3 activation, and downstream gasdermin E (GSDME) processing. Inhibition of caspase-3 significantly attenuated cell death, and caspase-8 inhibition rescued viability and prevented Bid cleavage, caspase-3 activation, and GSDME cleavage. These effects occurred in the context of enhanced endoplasmic reticulum stress, increased DNA damage with reduced DNA repair, and impaired Akt-driven survival signalling. ConclusionsOur findings establish that NFV synergizes with CDDP in killing Pt-resistant ovarian cancer cells by promoting a caspase-8-dependent apoptotic-to-secondary pyroptotic response, supporting further investigation of NFV as a potential drug to be repurposed to increase the efficacy of Pt-based therapy.

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.

Mutations in isocitrate dehydrogenase 1 (IDH1) drive the early stages of gliomagenesis while simultaneously imposing replication stress that creates targetable vulnerabilities. Using both in vitro and in vivo models, we show that inhibition of poly(ADP-ribose) glycohydrolase (PARG) induces a poly(ADP-ribose) (PAR)-dependent augmentation of radiosensitivity in IDH1-mutant glioma cells. Metabolic repletion of NAD+ fails to rescue this effect, indicating that the vulnerability cannot be explained solely by NAD+ depletion. Instead, PARG inhibition profoundly alters replication fork progression and S-phase kinetics in IDH1-mutant cells. Mechanistically, ionizing radiation preferentially activates replication fork-associated damage response proteins DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and X-ray repair cross-complementing protein 1 (XRCC1) in IDH1-mutant cells, a response partially reversed by pharmacologic inhibition of mutant IDH1. Importantly, pharmacologic inhibition of DNA-PKcs with AZD7648 during irradiation disrupts fork-associated repair signaling and markedly enhances cytotoxicity in IDH1-mutant glioma models. Together, these findings identify a PAR-dependent replication fork vulnerability that can be therapeutically exploited to selectively enhance radiosensitivity in IDH1-mutant gliomas. Statement of significanceIDH-mutant gliomas harbor intrinsic replication stress yet lack targeted radiosensitization strategies. We identify a PAR-dependent replication fork vulnerability in which disruption amplifies radiation cytotoxicity by deregulating S-phase fork signaling. Pharmacologic DNA-PKcs inhibition exploits this dependency, providing a genotype-selective approach to enhance radiotherapy in IDH-mutant glioma.

Cisplatin resistance remains a major barrier in head and neck squamous cell carcinoma (HNSCC) treatment. ATP-binding cassette (ABC) transporters contribute to chemoresistance by limiting intracellular drug accumulation. Bitter taste receptor 10 (T2R10) has been implicated in ABC transporter regulation, but its role in HNSCC remains undefined. HNSCC cell lines were treated with T2R10-agonist caffeine (100 or 200 M), cisplatin, or a combination, and viability was assessed by crystal violet assay. T2R10 promoter activity and expression following caffeine exposure were evaluated using a promoter-driven mCherry reporter and RT-qPCR. ABC transporter expression was measured after caffeine treatment and T2R10 gene (TAS2R10) knockdown or overexpression. Associations between tumor TAS2R10 expression and survival were assessed using TCGA data through GEPIA2. Caffeine enhanced the cisplatin-associated reduction in viability in a cell line- and concentration-dependent manner, with the strongest effect seen in UM-SCC47. A significant effect was observed in FaDu at 200 M of caffeine, and minimal response in RPMI 2650. RPMI 2650 cells and FaDu cells exhibited lower baseline TAS2R10 expression and RPMI 2650 cells did not demonstrate enhanced cisplatin sensitivity following caffeine treatment. Caffeine treatment increased TAS2R10 promoter activity and expression and was associated with decreased ABCG2 expression. TAS2R10 knockdown increased ABCG2 and ABCF1 expression, whereas TAS2R10 overexpression reduced ABCG2 and ABCC1 expression. High tumor TAS2R10 expression was associated with improved disease-free survival (log-rank p=0.0071; HR=0.61) but not overall survival. Caffeine enhances cisplatin sensitivity in selected HNSCC models. Caffeine exposure is associated with increased TAS2R10 expression and reduced expression of chemoresistance-associated transporters, particularly ABCG2.

BET bromodomain inhibitors block binding of BET family bromodomains 1 and 2 (BD1, BD2) to chromatin and have demonstrated clinical activity in NUT carcinoma (NC), a BRD-NUT fusion-driven cancer, but toxicity from BD1 inhibition has limited their effectiveness. We investigated whether selective inhibition of BRD4 bromodomain 2 (BD2) could retain antitumor activity while reducing toxicity. NC cells were uniquely sensitive to the novel BRD4-BD2 inhibitor DC-9476 and other BD2-selective inhibitors, which induced differentiation and growth arrest. A CRISPR knockout screen identified the BRD4-targeting E3 ligase SPOP as the top resistance hit. BD2 inhibition, but not BD1-selective or pan-BET inhibition, triggered SPOP-dependent proteasomal degradation of BRD4 and BRD4-NUT; SPOP loss prevented degradation and largely rescued BD2 inhibitor-induced differentiation and growth arrest. Unexpectedly, BRD4 and BRD4-NUT remained chromatin-associated during BD2 inhibition, whereas BD1 or pan-BET inhibition displaced them. Together with evidence that ectopic BRD4-NUT expression sensitizes BRD4 to degradation, these findings support a model in which BRD4-NUT megadomains create a high-density, degradation-competent SPOP substrate pool of BRD4 and BRD4-NUT upon BD2 inhibition, whereas pan-BET inhibition disperses this substrate and limits efficient degradation. In preclinical NC models, BD2-selective inhibition achieved greater tumor growth inhibition and survival benefit than pan-BET inhibition, revealing a therapeutic vulnerability.

BackgroundMedulloblastoma is the most common malignant pediatric brain tumor. Although advances in conventional therapies have improved survival over the years, acquired drug resistance remains a major barrier to durable cure. As dysregulation of epigenetic mechanisms is increasingly recognized as a driver of medulloblastoma pathogenesis and therapeutic adaptation, targeting epigenetic vulnerabilities represents a promising strategy to overcome treatment resistance. MethodsWe generated vincristine-resistant medulloblastoma cell line models and performed chemical screening to identify therapeutically targetable vulnerabilities. Candidate hits were validated using transcriptomic analyses, chromatin immunoprecipitation, and CRISPR-mediated genetic ablation to define the molecular mechanisms underlying drug sensitivity. ResultsChemical screening identified multiple active epigenetic compound classes capable of resensitizing vincristine-resistant medulloblastoma cells, including histone methyltransferase inhibitors, histone deacetylase inhibitors, and bromodomain inhibitors. Among these, the CBP/p300 bromodomain inhibitor SGC-CBP30 emerged as the most potent sensitizer to vincristine. Transcriptomic profiling revealed that, while ABCB1 was among the most highly upregulated genes in resistant cells, SGC-CBP30 treatment selectively downregulated ABCC3 and ABCA4, an effect not observed in parental cells. Mechanistically, chromatin immunoprecipitation demonstrated enrichment of p300 and H3K27ac at the ABCC3 and ABCA4 promoters in resistant cells, which was markedly reduced following SGC-CBP30 treatment. Consistent with these findings, genetic ablation of CREBBP or EP300 phenocopied the effects of pharmacological inhibition. Analysis of patient datasets further demonstrated elevated CREBBP, EP300, and ABCC3 expression in SHH MB, with positive correlations between ABCC3 and both CREBBP and EP300, supporting the clinical relevance of this regulatory axis. ConclusionsTogether, our findings demonstrate that CBP/p300 activity contributes to acquired vincristine-resistance in medulloblastoma. Targeting this axis represents a promising strategy to overcome drug resistance and enhance the efficacy of vincristine-based chemotherapy particularly in the context of relapsed or refractory disease. PLAIN ENGLISH SUMMARYMedulloblastoma is the most common cancerous brain tumor in children. Although many children respond well to the treatment, some tumors become resistant to chemotherapy, making them much harder to treat. Understanding why this resistance develops could lead to better treatment options for children whose cancer returns or no longer respond to therapy. In this study, we created laboratory models of medulloblastoma that had become resistant to the chemotherapy drug vincristine. We then tested a collection of drugs to identify compounds, which would restore the cancer cells sensitivity to treatment. We have discovered that several drugs were effective, with one compound, called SGC-CBP30, showing particularly strong activity. We investigated how SGC-CBP30 works and found that it decreases the activity of genes that are linked to chemotherapy resistance. Using multiple complementary experimental approaches, we confirmed that this gene-regulating pathway plays an important role in helping medulloblastoma cells survive treatment. Our findings suggest that targeting this pathway could restore the effectiveness of chemotherapy in drug-resistant tumors. Although further research is needed before this approach can be used in patients, these results provide a promising foundation for developing new treatments for children with relapsed or treatment-resistant medulloblastoma.

Homologous recombination deficiency (HRD) is an actionable vulnerability found in a substantial fraction of human cancers, yet current HRD-directed therapies are limited by toxicity, incomplete responses, and acquired resistance. Many DNA-damaging agents were developed before DNA repair biomarkers were available, suggesting that abandoned agents may harbor previously unrecognized genotype-selective activity. Here, through a focused screen of DNA-damaging agents in isogenic homologous recombination-proficient and -deficient models, we identify CB1954, a decades-old nitrobenzamide aziridine prodrug, as highly selective for BRCA2-deficient tumor cells. CB1954 forms DNA interstrand crosslinks independent of HR status, but selectively induces DNA-damage signaling, apoptosis, and loss of clonogenic survival in HR-deficient cells. Targeted DDR CRISPR screening and isogenic validation define a distinct repair dependency for the Fanconi anemia and homologous recombination pathways, with limited dependence on mismatch repair or nucleotide excision repair. Genetic and pharmacologic perturbation of NQO2, the bioactivating enzyme for CB1954, reveals a bifurcated mechanism in which NQO2-dependent activation selectively contributes to HRD cytotoxicity, while aziridine-dependent lesions likely account for residual activity in HR-proficient cells. CB1954 exhibits favorable preclinical pharmacokinetic properties and genotype-dependent antitumor activity in BRCA2-deficient xenografts. These findings reposition CB1954 as a historically overlooked HRD-selective agent and demonstrate that biomarker-guided profiling of DNA-damaging agents can uncover new opportunities for precision oncology.

Myeloproliferative neoplasms (MPN) are chronic hematologic malignancies characterized by clonal myeloid expansion, inflammation, oxidative stress, and progressive bone marrow (BM) remodeling that may culminate in fibrosis and secondary acute leukemia. Here, we evaluated the therapeutic efficacy and the underlying mechanisms of melatonin (MT) and liposomal melatonin (nano-MT) in preclinical MPN models. MT selectively inhibited clonogenic growth of patient-derived peripheral blood mononuclear cells and induced pluripotent stem cell-derived CD34 hematopoietic stem and progenitor cells in comparison to healthy controls. This effect was associated with increased apoptosis, reduced reactive oxygen species (ROS), and decreased glucose uptake, independently of MT receptor signaling. Transcriptomic profiling of primary MPN CD34 cells revealed suppression of MYC targets, G2M checkpoint signaling, ROS, and glycolysis pathways. In co-culture models, MT reduced stromal -smooth muscle actin and phosphorylated SMAD2/3, indicating inhibition of TGF-{beta}-driven mesenchymal stromal cell-to-myofibroblast formation. In tamoxifen-inducible SclCreER;JAK2V617F mice, nano-MT achieved efficient spleen and BM targeting. Therapeutically, nano-MT reduced erythrocytosis, myeloid progenitor expansion, and BM IL-1{beta} levels. Longitudinal micro-computed tomography and histological analyses demonstrated normalization of BM architecture, reduced osteosclerotic remodeling and splenomegaly, decreased reticulin deposition and megakaryocyte numbers. In a dose-escalation study, nano-MT restored erythrocyte, hematocrit, and platelet counts and normalized megakaryocyte-erythroid progenitors. Combination treatment with ruxolitinib further reduced leukocytosis, neutrophilia, and monocytosis. Collectively, these findings demonstrate that (nano-)MT attenuates MPN and BM remodeling by targeting metabolic, inflammatory, and fibrotic pathways. This study provides the first evidence for a therapeutic benefit of nano-MT in MPN and establishes a rationale for further translational evaluation.

Polo-like kinase 1 (PLK1), a key regulatory protein controlling entry into and passage through mitosis, has been targeted through its kinase domain (KD) with mixed clinical success. Inhibition through the Polo-box domain (PBD) is a viable alternative through targeting the sub-cellular localization and kinase activity of PLK1. Novel non-peptidic compounds, termed abbapolins, were discovered through the REPLACE strategy and have been lead optimized through structure-based strategies and screening analogs in the NCI-60 tumor cell panel. Proteomic analysis revealed a correlation between abbapolin activity and PLK1 protein levels in the cell lines part of the NCI-60. Prostate cell lines were identified as among the most sensitive and led to further detailed studies of their activity in prostate cancer models. Compounds were evaluated for their pharmacokinetic properties, and in vivo efficacy, and results showed significant antitumor xenograft activity with no observable gross toxicity. Treated tumors were analyzed for loss of PLK1, which was previously shown to be induced by abbapolin binding. Results obtained showed a significant degradation of PLK1 in abbapolin-treated vs untreated tumors, thereby confirming on-target action in vivo and revealing PLK1 levels as a potential pharmacodynamic marker. Lead compounds were shown to sensitize PC tumors resistant to androgen deprivation therapy paving the way for future combination studies in vivo. These data provide an alternative pathway for effective PLK1 therapeutics that avoid the reported problems of molecules targeting the KD, in vivo proof-of-concept for the REPLACE strategy and validation for targeting the PBD as an anti-tumor drug development strategy.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an immunosuppressive tumor microenvironment (TME) with pancreatic myofibroblasts (PMF) and macrophages being two prominent cell populations essentially impairing tumor responses to (immuno)therapies. L1 cell adhesion molecule (L1CAM) is upregulated in PDAC cells in primary and metastatic tissues and associated with tumor progression and therapy resistance. Using L1CAM as tumor-associated antigen, two bispecific antibodies (bsAB) targeting L1CAM and CD3 were developed in the IgG-(L)-ScFv format and their anti-tumorigenic activity was investigated in different preclinical PDAC models. In 2D models, both L1-bsAB exerted L1CAM-specific anti-PDAC cell activity when co-cultured with activated CD8+ T cells. Strong anti-PDAC cell effects along with elevated release of T cell effector molecules were also observed upon co-culture with peripheral blood mononuclear cells (PMBC) from healthy donors and PDAC patients. Of note, both L1-bsAB were also effective in 3D PDAC cell spheroids and neither impaired by PMF nor macrophages. Finally, application of L1-bsAB on organotypic tissue slice cultures from PDAC tissues comprising the entire complex TME also induced PDAC cell apoptosis and release of T cell effector molecules. Overall, our results highlight relevant anti-PDAC cell activity of L1-bsAB in immunosuppressive contexts supporting their potential as immunotherapeutic strategy for PDAC.