Molecular Cancer Therapeutics

PurposeMost prostate cancer (PCa) patients treated with androgen receptor (AR)-signaling inhibitors develop therapeutic resistance due to restoration of AR-functionality. Thus, there is a critical need for novel treatment approaches. Here we investigate the theranostic potential of hu5A10, a humanized monoclonal antibody specifically targeting free prostate-specific antigen (KLK3). Experimental DesignLNCaP-AR xenografts (NSG mice) and KLK3_Hi-Myc transgenic mice were imaged with 89Zr- or treated with 90Y- or 225Ac-labeled hu5A10; biodistribution and subcellular localization were analyzed by gamma-counting, positron emission tomography (PET), autoradiography and microscopy. Therapeutic efficacy of [225Ac]hu5A10 and [90Y]hu5A10 in LNCaP-AR tumors was assessed by tumor volume measurements, time to nadir (TTN), time to progression (TTP), and survival. Pharmacokinetics of [89Zr]hu5A10 in non-human primates (NHP) were determined using PET. ResultsBiodistribution of radiolabeled hu5A10-constructs was comparable in different mouse models. Specific tumor uptake increased over time and correlated with PSA expression. Treatment with [90Y]/[225Ac]hu5A10 effectively reduced tumor burden and prolonged survival (p[&le;]0.0054). Effects of [90Y]hu5A10 were more immediate than [225Ac]hu5A10 (TTN, p<0.0001) but less sustained (TTP, p<0.0001). Complete responses were observed in 7/18 [225Ac]hu5A10 and 1/9 mice [90Y]hu5A10. Pharmacokinetics of [89Zr]hu5A10 were consistent between NHPs and comparable to those in mice. [89Zr]hu5A10-PET visualized the NHP-prostate over the 2-week observation period. ConclusionsWe present a complete preclinical evaluation of radiolabeled hu5A10 in mouse PCa models and NHPs, and establish hu5A10 as a new theranostic agent that allows highly specific and effective downstream targeting of AR in PSA-expressing tissue. Our data support the clinical translation of radiolabeled hu5A10 for treating PCa.

Six transmembrane epithelial antigen of the prostate 1 (STEAP1) is a compelling tumor-associated cell surface antigen for therapeutic targeting in solid tumors. We identified broad expression of STEAP1 (87% positive) in lethal metastatic prostate cancer, even more so than prostate-specific membrane antigen (PSMA, 60% positive) which is a clinically established diagnostic and therapeutic target. Second-generation chimeric antigen receptor (CAR) T cells were engineered for reactivity against STEAP1 and demonstrated substantial antitumor activity in metastatic human prostate cancer models in immunodeficient mice. Adoptive transfer of STEAP1 CAR T cells was associated with prolonged peripheral persistence and either disease eradication or substantial tumor growth inhibition with progressive disease demonstrating antigen loss. As STEAP1 CAR T cells were also highly active in antigen density conditions as low as [~]1,500 molecules/cell, we generated a human STEAP1 (hSTEAP1) knock-in (KI) mouse to evaluate the potential for on-target off-tumor toxicities. hSTEAP1-KI mice demonstrated a pattern of systemic hSTEAP1 expression akin to that observed in humans with the greatest expression found in the prostate gland. Mouse-in-mouse studies of STEAP1 CAR T cell therapy in immunocompetent hSTEAP1-KI mice engrafted with disseminated mouse prostate cancer showed preliminary safety without evidence of gross toxicity, cytokine storm, or architectural disruption and increased T cell infiltration at sites of systemic hSTEAP1 expression. Tumor responses and extension of survival were appreciated but antigen loss was identified in recurrent and progressive disease. In summary, we report the extent of STEAP1 expression in treatment-refractory metastatic prostate cancer, the generation of a STEAP1 CAR T cell therapy with promising potency and safety in preclinical studies of advanced prostate cancer, and antigen escape as a mechanism of resistance to effective STEAP1 CAR T cell therapy.

Here, we describe a novel pan-RAS inhibitor, ADT-007, that potently inhibited the growth of RAS mutant cancer cells irrespective of the RAS mutation or isozyme. RASWT cancer cells with GTP-activated RAS from upstream mutations were equally sensitive. Conversely, RASWT cancer cells harboring downstream BRAF mutations and normal cells were essentially insensitive to ADT-007. Sensitivity of cancer cells to ADT-007 required activated RAS and dependence on RAS for proliferation, while insensitivity was attributed to metabolic deactivation by UDP-glucuronosyltransferases expressed in RASWT and normal cells but repressed in RAS mutant cancer cells. ADT-007 binds nucleotide-free RAS to block GTP activation of effector interactions and MAPK/AKT signaling, resulting in mitotic arrest and apoptosis. ADT-007 displayed unique advantages over mutant-specific KRAS and pan-KRAS inhibitors, as well as other pan-RAS inhibitors that could impact in vivo antitumor efficacy by escaping compensatory mechanisms leading to resistance. Local administration of ADT-007 showed robust antitumor activity in syngeneic immune-competent and xenogeneic immune-deficient mouse models of colorectal and pancreatic cancer. The antitumor activity of ADT-007 was associated with the suppression of MAPK signaling and activation of innate and adaptive immunity in the tumor immune microenvironment. Oral administration of ADT-007 prodrug also inhibited tumor growth, supporting further development of this novel class of pan-RAS inhibitors for RAS-driven cancers. SIGNIFICANCEADT-007 has unique pharmacological properties with distinct advantages over other RAS inhibitors by circumventing resistance and activating antitumor immunity. ADT-007 prodrugs and analogs with oral bioavailability warrant further development for RAS-driven cancers.

Metastatic castration-resistant prostate cancer (mCRPC) is an aggressive subtype of prostate cancer (PC) without curative treatments. Antibody-drug conjugates (ADCs) emerged as promising cancer therapeutics that selectively deliver cytotoxic agents (payloads) to the tumors. Although ADCs have been successfully applied in the treatment of hematological and solid tumors, ADC monotherapy has not demonstrated durable responses in mCRPC and the mechanisms of PC resistance to ADCs have not been thoroughly investigated. Our study aimed to improve ADC efficacy using a new integrated approach for custom ADC design and multiplexing. To nominate rational combinations of ADC targets and ADC payloads, we (1) examined protein co-expression of three clinically relevant surface antigens-- B7 homolog 3 (B7-H3), prostate specific membrane antigen (PSMA), and six-transmembrane epithelial antigen of prostate-1 (STEAP1)--in a series of human mCRPC samples and (2) screened established ADC payloads and their combinations in mCRPC cell lines with different phenotypes. We identified synergistic interactions between DNA-damaging payloads and Bcl-xL inhibitor A-1331852 as well as their coordinated induction of the intrinsic apoptosis pathway. The functional relevance of isolated p53 loss and impaired PC responses to three genotoxic ADCs (B7-H3-seco-DUBA, PSMA-SG3249, and STEAP1-DXd) and their combinations with A-1331852 was established using genetic knockout models. Lastly, we found enhanced in vivo antitumor activity in mCRPC by combining the clinically relevant agents B7-H3-seco-DUBA (vobramitamab duocarmazine) and A-1331852. Collectively, our findings provide rationale for the development of ADC therapies combining genotoxic payloads with Bcl-xL inhibitors for mCRPC. SignificanceB7-H3, PSMA, and STEAP1 targeted ADC therapies combining genotoxic payloads with Bcl-xL inhibitors induce p53-dependant apoptotic cell death in mCRPC, providing a clinically viable strategy for the treatment of advanced prostate cancer.

PurposeRadioligand therapy (RLT) is relatively unexplored in metastatic castration resistant prostate cancer (mCRPC), with much of the focus having been on bone seeking radionuclides and PSMA-directed RLT. Herein, we evaluated if CUB domain containing protein 1 (CDCP1) can be exploited to treat mCRPC with RLT, particularly for subsets like small cell neuroendocrine prostate cancer (SCNC) that would not be expected to respond to current options. Experimental DesignCDCP1 mRNA levels were evaluated in the RNA-seq data from 119 recent mCRPC biopsies. Protein expression was assessed in twelve SCNC and adenocarcinoma patient derived xenografts. Saturation binding assays were performed with 4A06, a recombinant human antibody that targets the CDCP1 ectodomain. The feasibility of imaging and treating mCRPC in vivo was tested with 89Zr-4A06 and 177Lu-4A06. ResultsCDCP1 mRNA expression was observed in over 90% of mCRPC biopsies, including SCNC and in adenocarcinoma with low FOLH1 (PSMA) levels. A modest anticorrelation was observed between CDCP1 and PTEN. Overall survival was not significantly different based on CDCP1 mRNA levels, regardless of PTEN status. Full length and/or cleaved CDCP1 was expressed in ten of twelve PDX samples. Bmax values of ~22,000 and ~6,200 fmol/mg were calculated for two human prostate cancer cell lines. Five prostate cancer models were readily detected in vivo with 89Zr-4A06. 177Lu-4A06 significantly suppressed the growth of DU145 tumors compared to control. ConclusionsThe antitumor data and the overexpression of CDCP1 reported herein provide the first evidence promoting CDCP1 directed RLT as a treatment strategy for mCRPC. Statement of Translational RelevanceNew targets for RLT are needed to address the subset of mCRPC that cannot be treated with bone seeking radionuclides or PSMA directed RLT. We report herein the first data credentialing CDCP1 as a target for mCRPC, in both adenocarcinoma and neuroendocrine subtypes. Combined with low expression in normal human tissues, these data provide a compelling scientific rationale for testing CDCP1 directed RLT clinically in mCRPC patients alone or in combination with other systemic therapies.

Somatostatin receptor 2 (SSTR2) is highly expressed in neuroendocrine tumors including small cell lung cancer (SCLC) and represents a validated target for peptide receptor radionuclide therapy. The SSTR2 agonist [177Lu]Lu-DOTA-TATE is clinically approved, however, treatment resistance and relapse occur. The SSTR2 antagonist SSO110 (DOTA-JR11, OPS201) demonstrates higher tumor uptake and longer retention than DOTA-TATE both pre-clinically and clinically. We performed a systemic head-to-head comparison of SSO110 labeled with various radionuclides of distinct emission characteristics to identify the optimal radionuclide for SSO110 and to compare antagonist with agonist performance. MethodsSSO110 was radiolabeled with 177Lu, 161Tb, 212Pb, and 225Ac. Biodistribution was assessed in AR42J and NCI-H69 xenograft models. Therapeutic efficacy of single and fractionated [212Pb]Pb-SSO110 was compared with [177Lu]Lu-SSO110 in NCI-H69 tumors. Single-dose efficacy of 225Ac-, 161Tb-, and 177Lu-labeled SSO110 was evaluated in both models. [{superscript 2}{superscript 2}Ac]Ac-DOTA-TATE served as agonist comparator. Tumor growth, survival, safety parameters, and tumor absorbed doses were analyzed. ResultsAll SSO110 radioconjugates demonstrated comparable biodistribution with high tumor uptake and favorable tumor-to-kidney ratios. In NCI-H69 tumors, [212Pb]Pb-SSO110 induced dose-dependent tumor growth delay but did not improve anti-tumor efficacy compared with [177Lu]u-SSO110 under single or fractionated regimens. [161Tb]Tb-SSO110 showed efficacy comparable to [177Lu]Lu-SSO110 in NCI-H69 model and significantly improved tumor growth delay in high-SSTR2-expressing AR42J tumors. Across both models, [225Ac]Ac-SSO110 demonstrated the highest therapeutic potency, inducing durable tumor regression and 100% survival at clinically relevant activities. [225Ac]Ac-SSO110 also outperformed the agonist comparator [225Ac]Ac-DOTA-TATE. Dosimetry analysis revealed a 63-fold higher tumor absorbed dose per injected administered activity for [225Ac]Ac-SSO110 compared with [212Pb]Pb-SSO110. All treatments were well tolerated without significant renal or hepatic toxicity. ConclusionTherapeutic efficacy of SSTR2-targeted peptide receptor radionuclide therapy appears to benefit from alignment between radionuclide physical half-life and ligand tumor residence time. Among the radionuclides evaluated, [225Ac]Ac-SSO110 demonstrated the most pronounced and durable anti-tumor efficacy, outperforming [161Tb]Tb-SSO110, [177Lu]Lu-SSO110, and the short-lived -emitter [212Pb]Pb-SSO110. These findings support clinical investigation of [225Ac]Ac-SSO110 in SSTR2-positive malignancies.

Radiopharmaceutical therapy is changing the standard of care in prostate cancer (PCa) and other malignancies. We previously reported high CD46 expression in PCa and developed an antibody-drug conjugate and immunoPET agent based on the YS5 antibody, which targets a tumor-selective CD46 epitope. Here, we present the preparation, preclinical efficacy, and toxicity evaluation of [225Ac]DOTA-YS5, a radioimmunotherapy agent based on the YS5 antibody. Our radiolabeled antibody retains binding efficacy and shows a high tumor to background ratio in PCa xenografts. Furthermore, we show that radiolabeled antibody was able to suppress the growth of cell-derived and patient-derived xenografts, including PSMA-positive and deficient models. Nephrotoxicity, not seen at low radioactive doses, is evident at higher radioactivity dose levels, likely due to redistribution of daughter isotope 213Bi. Overall, this preclinical study confirms that [225Ac]DOTA-YS5 is a highly effective treatment and suggests feasibility for clinical translation of CD46 targeted radioligand therapy in PCa.

BackgroundFluoropyrimidines, specifically 5-fluorouracil (5-FU), remain the cornerstone of colorectal cancer (CRC) therapy. However, intrinsic and acquired resistance, alongside dose-limiting systemic toxicities, often result in treatment failure and disease relapse. There is a pressing clinical need for next-generation fluoropyrimidines that can retain the antitumor activity in 5-FU-refractory CRC models while maintaining a favorable safety profile. MethodsWe evaluated the antitumor efficacy of CF10, a novel polymeric fluoropyrimidine designed for the sustained delivery of FdUMP, against equimolar 5-FU. We utilized a diverse panel of six patient-derived CRC organoid (PDO) models to assess 3D growth inhibition under both normoxic ([~]20% O2) and physioxic (5% O2) conditions. Mechanisms of action were investigated via {gamma}H2AX signaling (DNA damage), Annexin V/PI flow cytometry (death kinetics), and ALDEFLUOR assays (stem-like populations). Functional suppression of metastasis-associated phenotypes was evaluated using 3D Matrigel invasion assays. Finally, the therapeutic index and overall survival were validated in vivo using two independent patient-cell-derived xenograft (PCDX) models (TX-CC-199 and TX-CC-201). ResultsCF10 demonstrated significantly greater suppression of organoid growth compared to equimolar 5-FU across all patient-derived lines, regardless of morphological heterogeneity or oxygen tension. In 3D invasion assays, CF10 achieved superior anti-invasive activity even at a 10-fold lower molar dose than 5-FU. This functional advantage was mirrored by a marked depletion of the ALDH-high stem-like subpopulation, which was largely recalcitrant to 5-FU. Mechanistically, CF10 induced intensified replication stress, DNA damage and repair signaling ({gamma}H2AX, Top1cc/pRPA32, FANCD2), and pushed the CRC to irreversible/terminal, PI-positive death states. In vivo, CF10 treatment resulted in profound tumor growth inhibition and a robust survival advantage in two patient cell-derived xenograft (PCDX) models (Log-rank P<0.01) without inducing systemic weight loss or noticeable toxicity. ConclusionsBy integrating 3D patient-derived modeling with in vivo validation, we demonstrate that CF10 effectively overcomes the biological and pharmacological limitations of 5-FU. CF10 targets the aggressive, invasive, and stem-like subpopulations of CRC that drive clinical relapses. These findings provide a compelling translational rationale for the clinical development of CF10 as a superior alternative to standard fluoropyrimidines in both treatment-naive and refractory CRC. Significance StatementDespite the foundational role of 5-fluorouracil (5-FU) in colorectal cancer (CRC) therapy, resistance and systemic toxicity remain major barriers to curative outcomes. This study identifies CF10, a novel polymeric fluoropyrimidine, as a superior alternative that overcomes 5-FU resistance in biologically diverse patient-derived organoids and xenograft models. Crucially, CF10 demonstrates a unique capacity to suppress the invasive, aldehyde dehydrogenase (ALDH)-high stem-like subpopulations that likely survive standard chemotherapy (5-FU) by maintaining efficacy under physiological oxygen levels and providing a significant survival advantage in vivo with improved tolerability. CF10 represents a promising translational candidate for the treatment of both treatment-naive and refractory CRC.

Antibody-drug conjugates (ADCs) are promising targeted cancer therapy; however, patient selection based solely on target antigen expression without consideration for cytotoxic payload vulnerabilities has plateaued clinical benefits. Biomarkers to capture patients who might benefit from specific ADCs have not been systematically determined for any cancer. We present a comprehensive therapeutic and biomarker analysis of a B7H3-ADC with pyrrolobenzodiazepine(PBD) payload in 26 treatment-resistant, metastatic prostate cancer(mPC) models. B7H3 is a tumor-specific surface protein widely expressed in mPC, and PBD is a DNA cross-linking agent. B7H3 expression was necessary but not sufficient for B7H3-PBD-ADC responsiveness. RB1 deficiency and/or replication stress, characteristics of poor prognosis, conferred sensitivity and were associated with complete tumor regression in both neuroendocrine (NEPC) and androgen receptor positive(ARPC) prostate cancer models, even with low B7H3 levels. Non-ARPC models, which are currently lacking efficacious treatment, demonstrated the highest replication stress and were most sensitive to treatment. In RB1 wild-type ARPC tumors, SLFN11 expression or select DNA repair mutations in SLFN11 non-expressors governed response. Significantly, wild-type TP53 predicted non-responsiveness (7/8 models). Overall, biomarker-focused selection of models led to high efficacy of in vivo treatment. These data enable a paradigm shift to biomarker-driven trial designs for maximizing clinical benefit of ADC therapies.

Current treatments for advanced prostate cancer (PCa) primarily target the androgen receptor (AR) pathway. However, the emergence of castration-resistant prostate cancer (CRPC) and resistance to AR pathway inhibitors (APSIs) remains ongoing challenges. Here, we present BSJ-5-63, a novel proteolysis-targeting chimera (PROTAC) targeting cyclin-dependent kinases (CDKs) CDK12, CDK7, and CDK9, offering a multi-pronged approach to CRPC therapy. BSJ-5-63 degrades CDK12, diminishing BRCA1 and BRCA2 expression and inducing a sustained "BRCAness" state. This sensitizes cancer cells to PARP inhibitors (PARPis) regardless of their homologous recombination repair (HRR) status. Furthermore, CDK7 and CDK9 degradation attenuates AR signaling, enhancing its therapeutic efficacy. Preclinical studies, including both in vitro and in vivo CRPC models, demonstrate that BSJ-5-63 exerts potent anti-tumor activity in both AR-positive and AR-negative setting. This study introduces BSJ-5-63 as a promising therapeutic agent that addresses both DNA repair and AR signaling mechanisms, with potential benefits for a board patient population.

CD4+ T helper cells are required for CD8+ killer T cells to suppress tumor growth. An orally-available small molecule surrogate of alpha-1 antitrypsin, Alphataxin, was previously demonstrated to elevate the numbers of circulating and tumor-infiltrating CD4+ T cells and to suppress kidney tumor growth in mice. To determine whether Alphataxin might be effective in other T cell-responsive cancers, mice orthotopically implanted with colon tumors were treated using Alphataxin and anti-PD-1 as monotherapies or in combination. Combination therapy significantly suppressed tumor growth (ORR = 37.5%) and increased tumor-infiltrating CD4+ T cells, CD8+ T cells, NK cells, M2 macrophages, and DC2 dendritic cells. Release of IFN-{gamma} by helper T cells in the tumor microenvironment appeared to contribute to the effectiveness of killer T cells in suppressing tumor growth. Toxicology studies in rats revealed no untoward effects. Alphataxin, to our knowledge the first and only drug developed to rapidly and sustainably increase the number of circulating and tumor-infiltrating CD4+ helper T cells, is a powerful therapeutic that provides long-term remission in T cell-responsive cancers in combination with anti-PD-1.

As colorectal cancer (CRC) remains a leading cause of cancer-related death, identifying therapeutic targets and approaches is essential to improve patient outcomes. The EGFR ligand epiregulin (EREG) is highly expressed in RAS wildtype and mutant CRC with minimal expression in normal tissues, making it an attractive target for antibody-drug conjugate (ADC) development. In this study, we produced and purified an EREG monoclonal antibody (mAb), H231, that had high specificity and affinity for human and mouse EREG. H231 also internalized to lysosomes, which is important for ADC payload release. ImmunoPET and ex vivo biodistribution studies showed significant tumor uptake of 89Zr-labeled H231 with minimal uptake in normal tissues. H231 was conjugated to either cleavable dipeptide or tripeptide chemical linkers attached to the DNA-alkylating payload duocarmycin DM, and cytotoxicity of EREG ADCs was assessed in a panel of CRC cell lines. EREG ADCs incorporating tripeptide linkers demonstrated the highest potency in EREG-expressing CRC cells irrespective of RAS mutations. Preclinical safety and efficacy studies showed EREG ADCs were well-tolerated, neutralized EGFR pathway activity, caused significant tumor growth inhibition or regression, and increased survival in CRC cell line and patient-derived xenograft models. These data suggest EREG is a promising target for the development of ADCs for treating CRC and other cancer types that express high levels of EREG. While the efficacy of clinically approved anti-EGFR mAbs are largely limited by RAS mutational status, EREG ADCs may show promise for both RAS mutant and wildtype patients, thus improving existing treatment options. Significance: EREG-targeting antibody-drug conjugates demonstrate acceptable safety and robust therapeutic efficacy in RAS mutant and wildtype colorectal cancer, suggesting their potential as an alternative to EGFR-targeted therapy to benefit a broader patient population.

Here, we evaluated in vivo antitumor activity, target engagement, selectivity, and tumor specificity of ADT-1004, an orally bioavailable prodrug of ADT-007 having highly potent and selective pan-RAS inhibitory activity. ADT-1004 strongly blocked tumor growth and RAS activation in mouse PDAC models without discernable toxicity. As evidence of target engagement and tumor specificity, ADT-1004 inhibited activated RAS and ERK phosphorylation in PDAC tumors at dosages approximately 10-fold below the maximum tolerated dose and without discernable toxicity. ADT-1004 inhibited ERK phosphorylation in PDAC tumors. In addition, ADT-1004 blocked tumor growth and ERK phosphorylation in PDX PDAC models with KRASG12D, KRASG12V, KRASG12C, or KRASG13Q mutations. ADT-1004 treatment increased CD4+ and CD8+ T cells in the TME consistent with exhaustion and increased MHCII+ M1 macrophage and dendritic cells. ADT-1004 demonstrated superior efficacy over sotorasib and adagrasib in tumor models involving human PDAC cells resistant to these KRASG12C inhibitors. As evidence of selectivity for tumors from PDAC cells with mutant KRAS, ADT-1004 did not impact the growth of tumors from RASWT PDAC cells. Displaying broad antitumor activity in multiple mouse models of PDAC, along with target engagement and selectivity at dosages that were well tolerated, ADT-1004 warrants further development. SignificanceADT-1004 displayed robust antitumor activity in aggressive and clinically relevant PDAC models with unique tumor specificity to block RAS activation and MAPK signaling in RAS mutant cells. As a pan-RAS inhibitor, ADT-1004 has broad activity and potential efficacy advantages over allele-specific KRAS inhibitors by averting resistance. These findings support clinical trials of ADT-1004 for KRAS mutant PDAC.

BackgroundTaxanes are the most active chemotherapy agents in metastatic castration resistant prostate cancer (mCRPC) patients, yet resistance almost invariably occurs representing an important clinical challenge. Taxane-platinum combinations have shown clinical benefit in a subset of patients but the mechanistic basis and biomarkers remain elusive. ObjectiveTo identify mechanisms and response biomarkers for the antitumor efficacy of taxane-platinum combinations in mCRPC. Design, setting, and participantsTranscriptomic data from a publicly available mCRPC dataset of taxane-exposed and naive patients was analysed to identify response biomarkers and emerging vulnerabilities. Functional and preclinical validation was performed in taxane resistant mCRPC cell lines and genetically engineered mouse models (GEMM). InterventionmCRPC cells were treated with docetaxel, cisplatin, carboplatin and the CXCR2 inhibitor, SB265610. Gain and loss of function in culture of CXCR2 was achieved by overexpression or siRNA-silencing. Preclinical assays in GEMM mice tested the anti-tumor efficacy of taxane-platinum combinations. Outcome measurements and statistical analysisProliferation, apoptosis and colony assays measured drug activity in vitro. Preclinical endpoints in mice included growth, survival and histopathology. Changes in CXCR2, BCL-2 and chemokines were analysed by RT-qPCR and Western Blot. Human expression data was analyzed using GSEA, hierarchical clustering and correlation studies. GraphPad Prism software, R-studio, were used for statistical and data analyses. Results and limitationsTranscriptomic data from taxane-exposed human mCRPC tumors correlates with a marked negative enrichment of apoptosis and inflammatory response pathways accompanied by a marked downregulation of CXCR2 and BCL-2. Mechanistically, we show that docetaxel treatment inhibits CXCR2 and that BCL-2 downregulation occurs as a downstream effect. Further, we demonstrated that taxane resistance is directly associated to CXCR2 expression and that targeting of CXCR2 sensitizes prostate cancer (PC) cells to cisplatin. Finally, taxane-platinum combinations in vivo are highly synergistic and previous exposure to taxanes sensitizes mCRPC tumors to second line cisplatin treatment. ConclusionsTogether our data identifies an acquired vulnerability in taxane treated mCRPC patients with potential predictive activity for platinum-based treatments. Patient summaryA subset of patients with aggressive and therapy resistant PC benefits from taxane-platinum combination chemotherapy however, we lack biomarkers and mechanistic basis about how that synergistic effect occurs. Here, using patient data and preclinical models, we found that taxanes reduce cancer cell scape mechanisms to chemotherapy-induced cell death, hence turning these cells more vulnerable to additional platinum treatment.

Mutant KRAS is present in over 90% of pancreatic as well as 30-40% of lung and colorectal cancers and is one of the most common oncogenic drivers. Despite decades of research and the recent emergence of isoform-specific KRASG12C-inhibitors, most mutant KRAS isoforms, including the ones frequently associated with pancreatic ductal adenocarcinoma (PDAC), cannot be targeted directly. Moreover, targeting single RAS downstream effectors induces adaptive mechanisms leading to tumor recurrence or resistance. We report here on the combined inhibition of SHP2, a non-receptor tyrosine phosphatase upstream of KRAS, and ERK, a serine/threonine kinase and a key molecule downstream of KRAS in PDAC. This combination shows synergistic anticancer activity in vitro, superior disruption of the MAPK pathway, and significantly increased apoptosis induction compared to single-agent treatments. In vivo, we demonstrate good tolerability and efficacy of the combination. Concurrent inhibition of SHP2 and ERK induces significant tumor regression in multiple PDAC mouse models. Finally, we show evidence that 18F-FDG PET scans can be used to detect and predict early drug responses in animal models. Based on these compelling results, we will investigate this drug combination in a clinical trial (SHERPA, SHP2 and ERK inhibition in pancreatic cancer, NCT04916236), enrolling patients with KRAS-mutant PDAC.

Broad-spectrum RAS inhibition holds the potential to benefit roughly a quarter of human cancer patients whose tumors are driven by RAS mutations. However, the impact of inhibiting RAS functions in normal tissues is not known. RMC-7977 is a highly selective inhibitor of the active (GTP-bound) forms of KRAS, HRAS, and NRAS, with affinity for both mutant and wild type (WT) variants. As >90% of human pancreatic ductal adenocarcinoma (PDAC) cases are driven by activating mutations in KRAS, we assessed the therapeutic potential of RMC-7977 in a comprehensive range of PDAC models, including human and murine cell lines, human patient-derived organoids, human PDAC explants, subcutaneous and orthotopic cell-line or patient derived xenografts, syngeneic allografts, and genetically engineered mouse models. We observed broad and pronounced anti-tumor activity across these models following direct RAS inhibition at doses and concentrations that were well-tolerated in vivo. Pharmacological analyses revealed divergent responses to RMC-7977 in tumor versus normal tissues. Treated tumors exhibited waves of apoptosis along with sustained proliferative arrest whereas normal tissues underwent only transient decreases in proliferation, with no evidence of apoptosis. Together, these data establish a strong preclinical rationale for the use of broad-spectrum RAS inhibition in the setting of PDAC.

As potent triggers of innate immunity, STING agonists hold promise as active immunotherapeutic agents for cancer treatment. Second-generation STING agonists, suitable for systemic delivery, are being investigated in preclinical research and have entered clinical trials. Here, the novel synthetic STING agonist, BI-1703880 (STINGa), which was designed for intravenous delivery, was investigated for anti-tumour and immunological effects. We show that STINGa activates the STING pathway and results in a transient and dose-dependent upregulation and secretion of interferons and proinflammatory cytokines in vitro and in vivo. We show that intravenous administration of repeated dosing with low-dose STINGa is well tolerated. We report that radiotherapy (RT) and STING agonism synergizes to generate innate immune cell and CD8+ T cell responses that control tumour growth. Anti-tumour activity induced by combined RT / STINGa was reduced in mice lacking a functional immune system. RT / STINGa combination treatment also initiated development of protective immune memory. RT / STINGa upregulated PD-L1, PD-1 and CTLA-4 in the tumour microenvironment. Our findings show that combining RT / STINGa with immune checkpoint inhibitors further increases therapeutic benefit. Our data confirm STING as a therapeutic target in cancer and support the clinical development of BI-1703880 STING agonist, thereby suggesting radiotherapy as a potential combination for enhancing anti-tumour efficacy.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by KRAS-driven oncogenic signaling and tumor growth. Blockade of the KRAS ERK-MAPK pathway via small molecule direct RAS inhibitors has shown clinical promise, but intrinsic and acquired resistance limit the efficacy of these inhibitors as single agents. To identify potential combination strategies, we first assessed the ability of dordaviprone/ONC201, an FDA-approved agent, to inhibit PDAC cell and organoid growth. We observed that ONC201 reduced the growth of a broad panel of KRAS-mutant PDAC cell lines, and that the expression of mitochondrial protease ClpP was required for this efficacy. Mechanistically, we observed that treatment with ONC201 led to inhibition of mitochondrial respiration, causing a compensatory increase in glycolysis. Furthermore, ONC201 caused ClpP-dependent activation of PI3K-AKT-mTOR signaling and concurrent PI3K and mTOR inhibition further enhanced ONC201 growth suppression. ONC201 demonstrated an additive effect when combined with a RAS(ON) multi-selective inhibitor RMC-7977 in PDAC cells and organoids. Finally, PDAC cell lines with acquired resistance to RMC-7977 or KEAP1 loss-driven resistance retained sensitivity to ONC201. We propose that concurrent treatment with ONC201 may delay onset of resistance to RAS inhibitor therapy. Statement of SignificanceClpP activation by dordaviprone/ONC201 suppressed PDAC cell growth and overcame resistance to the RAS(ON) multi-selective inhibitor RMC-7977, providing support for investigating this combination as a potential combination treatment for KRAS-mutant pancreatic cancer.

Aldehyde dehydrogenase 1A3 (ALDH1A3) increases tumor growth, metastasis, and chemoresistance in many solid tumors, including triple-negative breast cancer (TNBC), glioblastoma, melanoma, lung, and colon cancers, yet no clinically approved inhibitors exist. Here, we present CLM296, a novel and highly selective ALDH1A3 inhibitor designed to address this unmet need. CLM296 exhibits potent inhibition of ALDH1A3 activity in TNBC cells (half-maximal inhibitory concentration = 2 nM) with no off-target effects on the highly homologous ALDH1A1 isoform. RNA sequencing confirmed its specificity, demonstrating selective suppression of ALDH1A3-regulated gene expression only, and a lack of effect in control cells that have minimal ALDH1A3 expression. Transwell assays showed that CLM296 reduced the increased invasion of cells induced by ALDH1A3. Once daily dosing of 4mg/kg CLM296 in mice specifically reduced ALDH1A3-mediated gene expression in tumors and impeded ALDH1A3-driven tumor growth and lung metastasis in TNBC xenografts. There was no observed toxicity in the mice as evidenced by stable mouse body weights and no significant changes in blood creatinine and ALT levels. Pharmacokinetic studies of CLM296 revealed broad tissue distribution, including tumor, lung, liver, and brain. With oral administration the terminal elimination half-life of CLM296 exceeded 12 hours, resulting in sustained ALDH1A3-inhibiting concentrations beyond 24 hours. Together, these findings establish CLM296 as a potential first-in-class ALDH1A3 inhibitor with high selectivity for ALDH1A3, favorable pharmacokinetics, and a positive preclinical safety profile. CLM296 represents a promising therapeutic candidate to complement standard-of-care treatments in ALDH1A3+ cancers.

PurposeTo assess the antitumor efficacy, pharmacokinetics, and safety of NV103, a CD99-targeted liposomal irinotecan nanoparticle, in a preclinical Ewing sarcoma model. Experimental DesignNV103 is a CD99-antibody targeted irinotecan containing nanoparticle, engineered to selectively deliver irinotecan to CD99 expressing tumor cells. In vitro studies measured binding, internalization, and cytotoxic IC50 values in several Ewing sarcoma cell lines. In vivo, mice bearing xenografts derived from treatment-naive and chemoresistant Ewing lines were treated. NV103 was compared with free irinotecan, untargeted nanoparticles, and OnivydeTM at multiple dosages. Plasma pharmacokinetics of irinotecan and SN-38, biodistribution of the nanoparticles, and toxicity (body weight, organ function, and hematology) were assessed. ResultsNV103 bound selectively to tumor cells (>80x over control), was rapidly internalized, and showed enhanced potency in vitro (IC50 {approx} 3-4 nM at 0.5-1 h). In vivo, biweekly dosing at 5 mg/kg resulted in full tumor regression sustained for 140 days, even after stopping treatment at day 70. Effective suppression and survival benefit were observed at doses as low as 1 mg/kg; the ED50 was estimated to be between 1-2.5 mg/kg versus 50mg/kg for free irinotecan. In a chemoresistant Ewing tumor cell line, NV103 induced similar tumor-free remission. Pharmacokinetics revealed prolonged and elevated plasma levels of irinotecan with NV103 versus free drug. No systemic toxicity was detected at doses of 10 mg/kg. Biodistribution showed tumor-preferential accumulation. ConclusionsNV103 displays potent and durable antitumor activity in Ewing sarcoma at low doses with no toxicity and favorable pharmacokinetics. These findings support further development for clinical translation. Translational RelevanceAlthough Irinotecan has shown activity against Ewing sarcoma, its clinical utility is limited by systemic toxicity and poor tumor selectivity. NV103, a CD99-targeted nanoparticle formulation of irinotecan delivers irinotecan selectively to Ewing sarcoma cells. CD99 is a surface antigen that is highly expressed in Ewing sarcoma cells but largely absent from irinotecan-sensitive organs like liver, kidney, and bone marrow. In preclinical xenograft models, NV103 induced complete and durable tumor regression at doses >10 fold lower than those of free irinotecan or untargeted nanoparticles, with no detectable systemic toxicity. These findings suggest that NV103 is a promising translational therapeutic agent that enhances the therapeutic index of irinotecan and other cytotoxic agents. This platform offers a broadly adaptable approach to tumor-specific drug delivery, which could significantly improve treatment outcomes and reduce long-term toxicity in children and young adults with Ewing sarcoma and other solid tumors.