Temozolomide (TMZ) is an established therapy for gliomas with silencing of the DNA repair gene O6-methylguanine-DNA methyltransferase (MGMT) but is hindered by the frequent development of resistance via loss of mismatch repair (MMR) proteins. To overcome this resistance, a novel 2-fluoroethylating agent, KL-50, was designed to generate toxic DNA interstrand cross-links specifically in MGMT-deficient cells, bypassing MMR. KL-50 has shown efficacy in preclinical MGMT-silenced, TMZ-resistant glioma models. MGMT loss also occurs in a wide range of nonglioma cancers, but the ability of KL-50 to effectively treat these tumors with minimal off-target toxicity remains untested. In this study, we utilize a large-scale cell-line screen, focused panels of cancer cell lines, and tumor xenograft mouse models, including patient-derived xenografts, to interrogate the therapeutic potential of KL-50 in treating intracranial and extracranial tumors. We find that KL-50 is highly efficacious in a wide range of MGMT-deficient human tumor models, including colon cancer, melanoma, and lung cancer in vivo models. In addition, we demonstrate that KL-50 is impervious to loss of MMR and remains effective in tumors with induced TMZ resistance. Finally, we determine that KL-50 possesses higher MGMT selectivity in vitro compared with chloroethylating agents and is associated with less in vivo systemic toxicity in preclinical models. These results establish KL-50 as the first agent to maintain strong selectivity for MGMT loss while inducing tumor toxicity by an MMR-independent mechanism and support further development of 2-fluoroethylating agents for use in a tumor type-agnostic, biomarker-based strategy targeting MGMT-deficient tumors.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers, and current therapeutics provide limited benefits to patients. Desmoplasia and the highly immunosuppressive tumor microenvironment (TME) play a huge role in PDAC being elusive to current therapeutics. Here, we strategically selected four transgenes to express from adenovirus vectors to target desmoplasia and the highly immunosuppressive TME in PDAC: interferon gamma (IFNγ), extracellular domain of transforming growth factor beta receptor 2 (sTGFβRII), interleukin 7 (IL7), and the extracellular domain of T-cell Immunoglobulin and Mucin domain-containing protein 3 (sTIM3). We used an immunocompetent syngeneic mouse model harboring tumors derived from KPC cells, a common mouse pancreatic cancer cell line, to test the anti-tumor effects of our transgene-expressing Ad vectors. We showed that all four transgenes elicited significant anti-tumor effects and shifted the tumors to a more immunogenic profile by increasing intratumoral expression of immune activation proteins and infiltration of anti-tumor immune cells. Furthermore, inhibition of desmoplasia was observed in three of four proteins. Of the four transgenes analyzed, IFNγ and sTGFβRII induced the greatest anti-tumor results and were combined in a single Ad vector. This dual-expressing Ad vector elicited the greatest anti-tumor effect, increase in immunogenicity, decrease in fibrosis, and showed abscopal effects. These results provide insight into how the analyzed transgenes can favorably alter the TME in pancreatic cancer patients and show encouraging potential when combined with other therapeutic regimens.

Acquired therapeutic resistance is a major problem in the treatment of advanced renal cell carcinoma (RCC). The receptor tyrosine kinase (RTK) c-Met is hyperactive in RCC and plays a major role in the survival of tumor cells during therapy-induced oxidative stress. Although c-Met/RTK inhibitor, cabozantinib (cabo), has demonstrated efficacy in the treatment of RCC, therapeutic resistance significantly restricts its effectiveness. Thus, new target molecules and combination therapies need to be explored. Among several cell cycle regulators, Polo-like kinase 1 (PLK1), which is overexpressed in RCC, can play a vital role in cancer progression. In this study, for the first time, we show that the activation of c-Met markedly promotes renal cancer cell growth and proliferation through PLK1. We found that a novel combination treatment with cabo and a PLK1 inhibitor (onvansertib) induces dual-phase cell cycle arrest at both G1 and G2/M checkpoints and impairs RCC cell migration. It induces tumor cell apoptosis by down-regulating the anti-oxidant transcription factor/cytoprotective molecule, Nrf2/HO-1, and inducing oxidative stress. Our results also showed that onvansertib can downregulate ABC transporters that are involved in drug resistance, and it resensitizes cabo-resistant RCC cells to apoptotic cell death. We further confirmed the effectiveness of the combination treatment through 3D spheroid culture as well as tumor xenograft models of RCC. Following combination treatment, there was increased drug-induced cytotoxicity, and markedly reduced tumor growth in vivo. Together, our pre-clinical study identifies that cabo + onvansertib is an effective combination treatment to overcome c-Met-induced therapeutic resistance in RCC with translational potential.

Cisplatin remains a crucial chemotherapeutic for head and neck squamous cell carcinoma (HNSCC), but its effectiveness is limited by intrinsic or acquired resistance driven largely by enhanced DNA crosslink repair. Through a combined screen of DNA crosslink repair and cisplatin sensitivity modulators, our current study identifies a strong synergy between cisplatin and microtubule-targeting agents (MTAs), including taxanes and colchicine. MTAs suppress DNA crosslink repair and overcome cisplatin resistance in HNSCC cells. Mechanistically, MTAs impair the resolution of cisplatin-induced lesions, consistent with reduced recruitment of the XPF-ERCC1 endonuclease required for DNA crosslink unhooking. MTAs also diminish homologous recombination, accompanied by reduced recruitment of RPA32 and RAD51 to DNA damage sites. Moreover, MTAs disrupt DNA damage checkpoint activation in response to cisplatin, permitting cell cycle transit through S-phase despite persistent DNA damage. Together, these effects drive enhanced DNA damage accumulation and apoptosis in HNSCC cells treated with the cisplatin-MTA combination. To further harness this synergy for therapeutic benefit, we employ poly(2-oxazoline)-based (POx) micelles to co-encapsulate paclitaxel and a hydrophobic cisplatin prodrug with two aliphatic hexane chains (C6CP). This nanoformulation was developed to improve drug solubility and release profiles, resulting in optimized pharmacokinetics and enhanced tumor delivery and retention. Indeed, in syngeneic HNSCC models, nanoparticle co-delivery of paclitaxel and cisplatin prodrug produced markedly superior antitumor efficacy compared to the free drug combination. Collectively, our findings define a mechanism-guided, nanoparticle-enabled therapeutic approach for sensitizing HNSCC to platinum-based chemotherapy through selective disruption of DNA damage responses.

Oncolytic viruses have emerged as a promising strategy in cancer immunotherapy and have been applied to the treatment of various tumors. We previously demonstrated that oncolytic adenoviruses engineered expressing tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) could induce apoptosis in tumor cells and modulate immune responses in the tumor microenvironment. To enhance targeting capability and antitumor immunity, the full-length TRAIL is intended to be replaced with a soluble TRAIL (sTRAIL). However, the sTRAIL is limited by structural instability and hepatic accumulation. In this study, we performed an optimized design based on the previously developed, more stable sTRAIL (FA1FT) by fusing it with a knobless fiber motif derived from fowl adenovirus type 1. Through structural analysis and genetic engineering, we developed a novel recombinant sTRAIL protein (mut-FA1TLST) that exhibited reduced liver tropism and enhanced antitumor activity both in vitro and in vivo. Furthermore, we incorporated this optimized sTRAIL into a capsid-modified oncolytic adenovirus, which demonstrated improved tumor targeting ability and antitumor efficacy. The recombinant oncolytic virus expressing the sTRAIL also promoted activation of CD4[+] T cells, CD8[+] T cells and DCs in the tumor microenvironment, thereby eliciting antitumor immune response. Overall, this study provides a promising recombinant sTRAIL candidate for preclinical and clinical applications and presents a novel oncolytic virus with potential for in vivo tumor immunotherapy.

Many cancers overexpress malic enzyme 1 (ME1), an enzyme essential in redox homeostasis through the generation of cytoplasmic NADPH. While ME1 overexpression is correlated with aggressive cancer phenotypes, little is known about the metabolic consequences of ME1 absence, recently identified as characteristic of synovial sarcoma. ACXT-3102 is a novel, dual-domain therapeutic produced by the conjugation of erastin, a small molecule xCT-inhibitor, and sigma-2 ligand, shown to induce ROS-dependent cell death in various cancer models. This study examines ME1 absence as a marker of sensitivity to xCT-inhibition in multiple malignancies and its role as a potential therapeutic marker for ACXT-3102 treatment. ME1-expressing and non-expressing tumor cell lines were identified and matched ME1 knockdown (KD) and overexpressing (OE) lines were created. Sensitivity to ACXT-3102 treatment was investigated in various tumor lines in vitro and in vivo. ME1 absence is identified in a subset of multiple malignancies. ME1 KD increases ME1-expressing tumor cell sensitivity to ACXT-3102, while ME1 re-introduction decreases xCT-inhibition sensitivity in ME1-null lines in in vitro and in vivo models. Compared to ME1-expressing cancers, ME1 absence increases tumor sensitivity to xCT inhibition. Our findings suggest ME1-null and low-expressing tumors constitute a distinct subset across multiple solid tumor types, representing a potential metabolic vulnerability. Given this differential sensitivity, ME1 deficiency may serve as a promising biomarker for selecting patients likely to benefit from xCT-targeting therapies. These results support further investigation into ME1 expression as a predictive tool for clinical trial stratification and of xCT-inhibition treatment strategies.

Rezatapopt (PC14586), a first-in-class Y220C-selective p53 reactivator, demonstrates single-agent clinical efficacy; however, combinatorial strategies may enhance clinical outcomes. Here, we evaluated rezatapopt in combination with standard-of-care agents (chemotherapy and bevacizumab), murine double minute 2 (MDM2) inhibitors, and a library of FDA-approved compounds. While chemotherapy, bevacizumab, and MDM2 inhibitors showed in vivo efficacy, a high-throughput screen identified the phosphoinositide 3-kinase (PI3K)/ Protein Kinase B (AKT)/mechanistic target of rapamycin (mTOR) and mitogen-activated protein kinase pathways as top synergistic candidates. Validation confirmed that PI3Kα inhibition synergized with rezatapopt to deepen apoptosis and tumor growth inhibition in xenograft models. Thus, PI3K/mTOR pathway inhibition may represent a clinically actionable, conserved p53-reactivation vulnerability across diverse histologies harboring a TP53 Y220C mutation.

Diffuse large B-cell lymphoma (DLBCL) is the most common and aggressive subtype of non-Hodgkin lymphoma (NHL), in which resistance to microtubule-targeting agents (MTAs) such as vincristine (VCR) remains a major therapeutic challenge. HEC1 (highly expressed in cancer 1) and NEK2 (NIMA-related kinase 2) are mitotic regulators that cooperate to maintain spindle integrity. Although HEC1 inhibitors that disrupt the HEC1-NEK2 interaction have been proposed as alternatives to MTAs with reduced neurotoxicity, their therapeutic relevance in DLBCL remains unknown. Here, we show that HEC1 and NEK2 are concomitantly upregulated in DLBCL, and their aberrant expression is associated with poor prognosis across multiple patient cohorts. The promoters of HEC1 and NEK2 show extensive sequence homology, and both genes are co-regulated by a shared transcriptional network including cell cycle-related transcription factors (TFs), involving FOXM1, MYC, and E2F family members, highlighting the central role of NEK2 in mitotic regulation together with the NDC80 complex. The HEC1 inhibitor TAI-1, which disrupts the HEC1-NEK2 interaction, induced degradation of HEC1 and NEK2, promoted GSK3β-dependent phosphorylation and degradation of β-catenin, and significantly suppressed DLBCL growth through mitotic catastrophe-associated apoptosis. Notably, HEC1 and NEK2 were upregulated in VCR-resistant DLBCL cells, and TAI-1 treatment restored VCR sensitivity and showed synergistic effects in combination with VCR. Our findings identify the HEC1-NEK2 axis as a therapeutic vulnerability in DLBCL and support combining HEC1 inhibitors with MTA-based chemotherapy to achieve superior anti-DLBCL efficacy.

Pancreatic ductal adenocarcinoma (PDAC) stands to become the second most deadly cancer by 2030. Mutations in the small GTPase, KRAS, occur in over 90% of PDAC patients and drive signaling plasticity through phosphorylation cascades. Protein phosphatases are master regulators of signal transduction, yet the contribution of phosphatase dysregulation to mutant KRAS phenotypes is poorly understood. Protein phosphatase 2A (PP2A) inhibits KRAS effectors, placing this family of enzymes as key regulators of PDAC signaling. However, we previously demonstrated that pharmacological activation of PP2A elicits heterogeneous responses in PDAC cells, with some cell lines displaying augmented oncogenic signaling despite increased phosphatase activity. Here, we determined the impact of the specific PP2A subunit, B56a, on PDAC phenotypes using both genetic and pharmacological activation strategies in human PDAC cell lines and genetic mouse models. We demonstrate that B56a knockout reduces PDAC proliferation while exogenous expression exacerbates proliferative phenotypes. This increase in proliferation predominately occurs through epidermal growth factor receptor (EGFR), a critical signaling node associated with poor patient outcome. The activation of EGFR by PP2A-B56a is mediated through increased expression and processing of EGFR ligands, such as amphiregulin. In vivo, the genetic loss of Cip2a, an endogenous PP2A inhibitor, significantly increases EGFR activation and decreases overall survival. Finally, pharmacological activation of PP2A leads to increased EGFR signaling in tumors; however, EGFR inhibition mitigates this signaling and significantly decreases tumor growth compared to single-agent treatment alone. Together, these studies implicate a previously undescribed non-canonical role for PP2A-B56a in EGFR signaling that contributes to PDAC progression.

The GAS6/AXL pathway plays a crucial role in promoting tumor survival and therapy resistance in various cancers. However, its significance in acute lymphoblastic leukemia (ALL) and the potential for its therapeutic targeting remain incompletely characterized. We integrated transcriptomic analysis of the public dataset GSE76349 with functional experiments in ALL cell lines. Cabozantinib was evaluated as an AXL inhibitor, and its effects on apoptosis, cell cycle, and signaling pathways were assessed using CCK-8 assays, RNA sequencing, Western blot, and flow cytometry. The functional role of AXL was confirmed via shRNA-mediated knockdown. The synergistic effect of combining cabozantinib with the AKT inhibitor capivasertib was evaluated in vitro and in a murine xenograft model. Bioinformatic analysis identified 8 key genes, including GAS6, associated with chemotherapy resistance. Cabozantinib exhibited strong cytotoxicity in AXL-high ALL cells, inducing apoptosis and G2/M arrest. Compensatory AKT activation was identified as a novel resistance mechanism in MOLT4 cells. AXL knockdown attenuated cabozantinib induced AKT suppression and apoptosis. Combined AXL and AKT inhibition synergistically enhanced anti leukemic effects in vitro and significantly reduced tumor burden in vivo with minimal toxicity. Our findings demonstrate that compensatory AKT activation limits the efficacy of cabozantinib in ALL and establish the combination of cabozantinib and capivasertib as a promising therapeutic strategy for refractory ALL.

Immune checkpoint blockade (ICB) has transformed cancer therapy, yet many tumors remain refractory to treatment due to diverse immune evasion mechanisms. To uncover novel drivers of ICB resistance, we performed an in vivo CRISPR-Cas9 screen in mice with increasing levels of immune pressure. This screen identified phosphatidylserine (PS) externalization as a potent mediator of immune escape. Tumors with elevated surface PS exposure prevented reinvigoration of CD8+ T cells upon PD-1 blockade, which was associated with increased recruitment of immunosuppressive myeloid cells. Targeting of PS Synthase 1 (PTDSS1) or either of the scramblases, XKR8 or ANO6, sensitized tumors to ICB in vivo. These findings demonstrate that elevated PS exposure constitutes a mechanism of resistance to ICB, and that targeting regulators of PS externalization represents a promising strategy to enhance the efficacy of immunotherapy in refractory tumors.

Metastatic brain tumors are common in patients with triple-negative breast cancer (TNBC) and often lead to a poor prognosis. Trophoblast cell surface antigen 2 (TROP2) is overexpressed in TNBC cells and an attractive target molecule in breast cancer. Datopotamab deruxtecan (Dato-DXd) is an antibody-drug conjugate targeting TROP2 that selectively releases payloads in tumors. We evaluated the efficacy and tumor concentration of payload of Dato-DXd and sacituzumab govitecan (SG), another TROP2-directed antibody-drug conjugate, in an intracranial TNBC xenograft model. We established the intracranial tumor model by orthotopically transplanting luciferase-expressing TROP2-positive TNBC cells into the brains of nude mice. Dato-DXd intravenously administered at 10 mg/kg on day 1 significantly reduced tumor size compared with SG administered at 10 mg/kg (average -99.5% vs. -1.8%, p = 0.02). The median survival was 18 days (95% confidence interval [CI]: 14 to not assessed [NA] days) in the control group, 48 days (15 to NA days) in the SG group, and 83 days (49 to NA days) in the Dato-DXd group. The payload released from Dato-DXd was rarely detected in the plasma (DXd: 0.09 ng/mL at maximum [at 6h postdose]), while the released payload of SG in the plasma was much higher than Dato-DXd and gradually decreased (SN-38: 10.8 ng/mL at maximum [at 0h postdose]). Dato-DXd showed greater antitumor activity in the intracranial TNBC model with higher payload accumulation in tumors compared with SG. Further clinical evaluation of Dato-DXd in patients with brain metastasis from TNBC is warranted.

CEACAM1 homodimerization mediates tumor immune evasion, presenting a compelling target to overcome resistance to PD-1 blockade. However, defining a precise structural and pharmacological blueprint for CEACAM1 targeting has remained challenging. Here, we report the discovery and comprehensive preclinical characterization of MG1124, a highly specific, fully human monoclonal antibody targeting the CEACAM1 IgV domain. X-ray crystallographic analysis at a 1.8 Å resolution revealed that MG1124 binds the homodimerization interface with robust affinity (KD = 37.8 nM), physically disrupting both homophilic and heterophilic inhibitory interactions without cross-reacting with highly homologous CEACAM members. This targeted structural blockade effectively reversed CEACAM1-mediated suppression of T-cell receptor signaling, restoring ZAP70 phosphorylation and IL-2 production, while potently enhancing NK- and CD8[+] T-cell cytotoxicity against CEACAM1-high tumor cells. In vivo, MG1124 reprogrammed the tumor immune microenvironment by increasing effector cytokine production in human CEACAM1 knock-in mice. It also demonstrated robust tumor growth inhibition in humanized non-small cell lung cancer (NSCLC) patient-derived xenograft (PDX) models, synergizing profoundly with PD-1 blockade. Furthermore, multiplex immunofluorescence profiling of human NSCLC tissues confirmed robust CEACAM1 expression across tumor and immune compartments, reinforcing its translational validity. Finally, MG1124 exhibited a highly favorable, drug-like pharmacokinetic profile in cynomolgus monkeys, characterized by prolonged systemic exposure and low clearance. By defining the structural basis of CEACAM1 blockade and demonstrating translational efficacy, this study introduces MG1124 as a novel therapeutic antibody, providing a robust pharmacological foundation for cancer immunotherapy.

Immune checkpoint inhibitors (ICIs) have improved cancer outcomes; however, many patients fail to respond, highlighting the need for novel targets. HVEM (Herpes Virus Entry Mediator) is an immune regulator with both inhibitory and stimulatory functions, making it a promising therapeutic candidate. We have developed Anti-4CB1, a fully human monoclonal antibody (mAb) that selectively blocks HVEM interactions with BTLA and CD160. Its activity was evaluated in-vitro using human tumor-infiltrating lymphocytes (TILs), peripheral blood mononuclear cells (PBMCs), and M1 macrophages, as well as in ex-vivo patient-derived tumor samples and in-vivo transgenic and humanized mouse models. HVEM expression was also assessed in serum and tumor tissues. Anti-4CB1 enhanced T-cell activation and cytotoxicity, evidenced by increased tumor cell killing, upregulation of activation markers (41BB, CD107a), and elevated IFNγ and TNFα secretion. It also promoted macrophage-mediated phagocytosis. In ex-vivo analyses of 49 patient-derived tumor samples, Anti-4CB1 increased cytotoxicity in 28.5% of cases, including samples unresponsive to anti-PD1. In-vivo, Anti-4CB1 demonstrated significant anti-tumor activity as monotherapy and showed enhanced efficacy in combination with anti-PD1. Additionally, higher tumor HVEM expression correlated with improved response to checkpoint blockade, while elevated soluble HVEM levels were associated with reduced responsiveness to Anti-HVEM. Anti-4CB1 enhances both adaptive and innate anti-tumor immunity and shows activity in anti-PD1 resistant settings. These findings support its potential as a novel therapeutic agent and suggest HVEM as a predictive biomarker for immunotherapy response.