Molecular Cancer Therapeutics最新文献

Cancer-associated fibroblasts (CAF) are key components of the tumor microenvironment (TME) that promote tumor progression either directly through tumor-CAF interactions or indirectly by influencing tumor-infiltrating immune cells, thereby creating an immunosuppressive TME. High stromal signatures have been associated with reduced therapeutic efficacy and resistance to immune checkpoint blockades. Adipocyte enhancer-binding protein 1 (AEBP1) is predominantly expressed in myofibroblasts, and its expression is further increased in CAFs that produce the extracellular matrix. It has two isoforms: The extracellular isoform binds to collagen and promotes collagen remodeling, whereas the intracellular isoform modulates transcription and signaling. We observed the expression of both isoforms in primary human CAFs. Our data showed that the combined knockout (KO) of both AEBP1 isoforms via gene editing decreased CAF proliferation, collagen gel contractility, and CAF-mediated tumor cell proliferation in vitro. AEBP1 KO mouse fibroblasts demonstrated reduced activity in both in vitro assays and in vivo within a coimplantation mouse model. RNA sequencing revealed that AEBP1 KO downregulated the collagen biosynthesis and extracellular matrix organization-related pathways in mouse fibroblasts and human CAFs. Importantly, AEBP1 loss in fibroblasts led to significant alterations in tumor cell phenotypes, including a marked reduction of tumor cells exhibiting an epithelial-mesenchymal transition signature in vivo. Furthermore, AEBP1 KO in CAFs enhanced the anti-PD-1-induced effector T-cell function and the anti-PD-1 efficacy. Our findings indicate that AEBP1 plays a crucial role in regulating the function of CAFs within the TME. Targeting AEBP1 could be a promising strategy to inhibit the tumor-promoting activities of CAFs and to overcome resistance to anti-PD-1 immunotherapy.

TUB-010 is a next-generation antibody-drug conjugate (ADC) targeting CD30 expressed on various hematopoietic malignancies such as Hodgkin lymphoma. Among the therapeutic options for patients with relapsed and refractory CD30-positive cancers is brentuximab vedotin (Adcetris), a monomethyl auristatin E (MMAE)-delivering anti-CD30 ADC with a mean drug-to-antibody ratio of 4. Adcetris exhibits a high response rate at the cost of significant toxicities, likely driven by the payload MMAE and the instability of the maleimide conjugation chemistry. TUB-010 uses the same antibody and payload as Adcetris but is based on the Tub-tag conjugation strategy, which stably attaches MMAE to the hydrophilic Tub-tag peptides on the light chains via chemoenzymatic conjugation. This new technology enables the generation of a homogeneous and site-specific drug-to-antibody ratio 2 ADC with unique biophysical properties. TUB-010 demonstrates similar binding and lysosomal release characteristics as Adcetris, which translates into comparable in vitro cytotoxicity on CD30-positive cell lines when normalized to the MMAE concentration. Importantly, TUB-010 exhibits higher stability with negligible premature deconjugation in circulation and reduced aggregation, as well as lower nonspecific cytotoxicity on target-negative cells compared with Adcetris. As a consequence, TUB-010 induces superior tumor control compared with Adcetris when dosed at equal MMAE concentrations in vivo and also lower toxicity and higher tolerability in rodents and nonhuman primates. Taken together, TUB-010 is a novel, potential best-in-class anti-CD30 ADC with improved biophysical properties designed to deliver MMAE with higher precision and a wider therapeutic window than Adcetris using Tub-tag technology. Therefore, TUB-010 may increase the clinical benefit of anti-CD30 ADC therapies.

Histone deacetylase (HDAC) inhibitors have been considered as anti-leukemic agents but have shown poor efficacy in clinical trials. In this study, we investigated the immediate transcriptional response to the HDAC inhibitor SAHA (vorinostat) in healthy CD34+ blood stem/progenitor cells and myeloblasts from patients with primary acute myeloid leukemia (AML) carrying TET2 and NPM1 mutations. We found that although healthy CD34+ and AML cells differed substantially at the transcriptional level, they responded very similarly to 10-minute SAHA treatment. HDAC inhibition led to a global increase in histone acetylation; however, only 150 to 250 genes were upregulated. These were involved in oxidative stress, metabolism, chromatin regulation, cell cycle control, and cell death, and the vast majority was upregulated in both healthy and AML cells. Upregulated genes were more highly acetylated compared with average expressed genes and had higher levels of promoter-proximal paused RNA polymerase II (Pol II) before treatment. Upon HDAC inhibition, upregulated genes increased BRD4 occupancy the most and released paused Pol II into transcription elongation. Our results suggest that the immediate effect of HDAC inhibition is to trigger release of paused Pol II into elongation. We speculate that the similar transcriptional response in healthy and leukemic cells may contribute to the poor efficacy of HDAC inhibitors in patients with hematologic malignancies.

Diffuse intrinsic pontine glioma (DIPG) is a lethal pediatric brain tumor with limited therapeutic progress due to its infiltrative brainstem location, blood-brain barrier (BBB), and resistance to systemic agents. We present a novel strategy for targeted central nervous system (CNS) delivery of ATSP-7041, a stapled peptide dual HDM2/HDMX inhibitor, using the Minimally Invasive Nasal Depot (MIND) technique. In p53-wild-type, PPM1D-mutant DIPG neurospheres (BT869), ATSP-7041 exhibited ~125-fold greater anti-tumor activity than the HDM2-selective antagonist RG7388, consistent with elevated HDMX expression. MIND delivery in mice achieved sustained ATSP-7041 distribution across brain regions, including the pons, with peak levels at 72 hours and persistence for up to 14 days. In a patient-derived orthotopic xenograft (PDX) model of DIPG, a single MIND-administered ATSP-7041 depot reduced tumor burden and prolonged survival compared to controls. This feasibility study provides proof-of-concept for on-target p53 reactivation in DIPG using a BBB-penetrant dual HDM2/HDMX inhibitor delivered by the MIND platform. The findings support a translational path for ALRN-6924, the clinical analog of ATSP-7041, in DIPG and potentially other brain tumors that retain wild-type p53 but remain incurable due to drug resistance and restricted CNS access.

EGFR Exon20 insertions (Exon20Ins) constitute the third most common EGFR activating mutation in non-small cell lung cancer. We developed a semi-mechanistic pharmacodynamic (PD) model for irreversible inhibitors of EGFR Exon20Ins mutations by integrating kinetic data of proprietary compounds with a mechanistic description of EGFR turnover and phosphorylation to investigate the preclinical relationship between phosphorylated EGFR (phosEGFR) reduction and efficacy, and its translation to the clinical setting. In engineered NCI-H2073 cells hosting the Exon20 SVDIns mutation, EGFR turnover was studied via SILAC MS and phosEGFR time-course analysed via ELISA. Kinetic parameters were determined from a biochemical binding assay. These data were integrated into the model to describe phosEGFR inhibition in vitro and in vivo. Tumour volume data from xenograft studies were then used to quantify the relationship between phosEGFR inhibition and anti-tumour activity. We found that sustained >84% phosEGFR inhibition is required for tumour regression. Clinical phosEGFR simulations were generated for 2 proprietary inhibitors, providing an early estimation of their active human doses. We also explored clinical phosEGFR reduction induced by the 3rd generation TKI osimertinib, suggesting that limited target engagement may explain modest response achieved in EGFR Exon20Ins at the clinically investigated doses. The developed model is a valuable tool to understand the impact of kinetic characteristics on phosEGFR reduction and related efficacy, select a target engagement-based criterion for therapeutic dose predictions, and provide interpretation and insights on observed clinical efficacy of irreversible inhibitors in EGFR Exon20Ins.

Mitogen-activated protein kinase kinase (MEK) is a component of an important signaling pathway involved in the development and progression of pancreatic ductal adenocarcinoma (PDAC). However, MEK-targeted therapeutics are not effective, and therefore not indicated, for patients with PDAC. We have found that Annexin A8 (ANXA8) is involved in resistance to MEK inhibitor therapy in PDAC. Expression of ANXA8 was induced at both the mRNA and protein levels early by MEK inhibitor treatment in PDAC cells and the level of ANXA8 mRNA expression was inversely correlated with sensitivity to the MEK inhibitor. Furthermore, downregulation of ANXA8 enhanced the inhibitory effect of MEK inhibitor on PDAC cell proliferation, suggesting that ANXA8 could be a potential therapeutic target for PDAC. To achieve a therapeutic strategy targeting ANXA8, we have identified all-trans retinoic acid (ATRA) as a compound exerting ANXA8-inhibitory effects in PDAC cells. Combination of the MEK inhibitor and ATRA demonstrated additive anti-tumor effects in PDAC cells in vitro and in vivo. Immunohistochemical analysis revealed that ANXA8 was frequently upregulated in PDAC showing poor differentiation relative to PDAC with high or moderate differentiation. Furthermore, patients with ANXA8-positive PDAC were found to have a significantly poorer prognosis than those with ANXA8-negative PDAC. In summary, our findings suggest that ANXA8 plays a role in MEK inhibitor resistance in PDAC, and that a combination of MEK inhibition with ANXA8-targeted therapy could be a novel effective strategy for PDAC.

The ganglioside GD2 is an attractive cancer target due to its high expression in neuroblastoma and other solid tumors, with limited normal tissues distribution. Despite regulatory approvals of three anti-GD2 antibodies, clinical efficacy remains limited by neurotoxicity, suboptimal affinity for ADCC, and immunogenicity. Developing a highly effective, less toxic anti-GD2 agent remains an unmet need. In this study, a novel anti-GD2 murine antibody, CA450, was identified by immunizing mice with GD2 conjugated to KLH or Qβ virus-like particles (VLP), followed by phage display screening. The humanized version, hCA450-21, displayed higher cell-binding activity than ch14.18 and Hu3F8, along with excellent specificity and internalization capacity. To overcome the limitations of traditional anti-GD2 antibody therapy, hCA450-21 was engineered and conjugated to Exatecan to create an antibody-drug conjugate (ADC), which may reduce or avoid neurotoxicity by employing a mechanism distinct from antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The hCA450-21.1-LA-68B ADC demonstrated potent in vitro cytotoxicity against glioblastoma, melanoma, and breast cancer cell lines, and in vivo tumor growth inhibition in LN229 and SK-MEL-5 xenograft models. Toxicity studies in mice showed a favorable safety profile, with reduced neurotoxicity compared to naked antibody therapy by ch14.18-IgG1 and hCA450-21.1-IgG1. The crystal structure of the hCA450-21.1 Fab-GD2 complex was resolved at 1.69 Å, revealing unique hydrogen bonds and hydrophobic interactions that contribute to its high specificity and affinity. Overall, the novel hCA450-21.1-LA-68B ADC shows preclinical efficacy and reduced toxicity, particularly neurotoxicity, indicating potential as a safer and more effective therapy for GD2-positive pediatric and adult tumors.

Small cell lung cancer (SCLC) is a recalcitrant thoracic malignancy known for acquired chemoresistance, early metastatic spread, and poor overall survival. Lurbinectedin, a DNA minor groove alkylating agent, provides durable efficacy in a minority. Predictive biomarkers for lurbinectedin are needed. Patients with relapsed SCLC who received lurbinectedin (n=16) were classified by cycles received, including eight durable responders defined as ≥8 cycles (average, 14.75 cycles; median PFS, 9.8 months). Pretreatment specimens were analyzed by immunohistochemistry (IHC) for SLFN11 and tandem mass tag (TMT)-labeled expression proteomics. Top candidates were confirmed by IHC and functionally validated in SCLC cell lines. SLFN11 failed to predict lurbinectedin response (P = 0.40). Proteomics highlighted a primitive neuroendocrine pathway (ISL1, SOX5, SIX1, SIX4). ISL1 expression significantly correlated with lurbinectedin response (r = 0.65, P = 0.0351). IHC confirmed lurbinectedin reduced ISL1 post-treatment. Lurbinectedin preferentially induced DNA damage in ISL1 "high" SCLC (P <0.0001) without causing neuroendocrine subtype switching. RNA sequencing showed downregulation of ISL1, RBMS3, ASCL1, SOX5, SIX1, and upregulation of ATF3. ISL1 "high" SCLC demonstrated cellular dependency on ISL1; ISL1 knockdown reduced lurbinectedin sensitivity. L-MYC positively regulated ISL1, while ISL1 positively regulated ASCL1 and SOX5. This is the first comprehensive investigation of predictive biomarkers for lurbinectedin. Proteomics identified ISL1 as defining a novel SCLC subtype with enhanced lurbinectedin sensitivity. ISL1 serves as both a predictive biomarker and functional dependency, as evidenced by essentiality for cell survival and loss following treatment. Prospective studies using ISL1 as a predictive biomarker for lurbinectedin are planned.

Chromobox 2 (CBX2), a subunit of Polycomb Repressor Complex 1 (PRC1), is expressed in high-grade serous carcinoma. CBX2 inhibitory peptide (CBX2i) has demonstrated efficacy in a syngeneic mouse model, but has limitations. We sought to identify an alternative approach to CBX2 inhibition. A computational-based molecular docking screen was performed using the SelleckChem Bioactive library to identify inhibitors of CBX2. A similarity screen of top hits against the bound conformation of CBX2i pharmacophore model was performed in parallel. A series of in vitro validation studies evaluated the effect of alisertib on proliferation, a CBX2 target gene, and stemness. CBX2 knockdown cell lines and a syngeneic murine model were utilized to evaluate alisertib response in the context of CBX2 loss. Cell target engagement assay was performed. PRC1-activity was measured by H2AK119ub levels. Immune profiling of treated tumors defined the immune microenvironment. The computational-based screen identified 10 candidate compounds. In vitro validation narrowed compounds of interest to raltitrexed, alisertib, GTX-007, LY315920, and PD0325901. Ultra-low dilution assay demonstrated dramatic decrease in spheroid formation with alisertib, an aurora A kinase (AURKA) inhibitor. Good structural overlap was observed between CBX2i and alisertib. Cell target engagement assay confirmed alisertib selectivity for both aurora A kinase and CBX2. Loss of CBX2 attenuated alisertib efficacy in vitro and in vivo. Treatment with alisertib leads to decrease in H2AK119ub and shift in the immune tumor microenvironment. Alisertib efficacy in HGSC is dependent on functional CBX2 and cell target engagement confirms selectivity for CBX2, supporting that alisertib activity involves CBX2 inhibition.

PD-1 immune checkpoint inhibition (ICI) is ineffective in most cancer patients. However, combination therapy can improve response rates, with the checkpoint TIGIT being a particularly interesting candidate as it is expressed on tumor-infiltrating exhausted T and NK cells. TIGIT's primary ligand, PVR, is overexpressed in many cancers and both TIGIT and PVR correlate with poor prognosis. To therapeutically exploit this, we developed a novel therapeutic termed Dual Signaling Protein 502 (DSP502). DSP502 is composed of the extracellular domains of TIGIT and PD-1, each fused to human IgG1 Fc containing knob-in-hole mutations. DSP502 was designed to simultaneously block PVR/TIGIT and PD-L1/PD-1 by binding to cancer cell-expressed PVR and PD-L1. Moreover, the human IgG1 domain can recruit FcR-positive effector cells to further reactivate anticancer immunity. Treatment with DSP502 potentiated NK cell activation and boosted the anticancer cytotoxicity of peripheral blood mononuclear cells (PBMCs) and tumor-infiltrating lymphocytes (TILs) from NSCLC and metastatic colorectal cancer patients towards cancer cells expressing both PD-L1 and PVR. Transcriptomic analysis confirmed NSCLC as a potential target, showing co-expression of TIGIT and PD-1 on a high percentage of exhausted CD8+ T cells. Notably, treatment with DSP502 not only blocked checkpoint signaling but also preserved surface expression of the co-stimulatory PVR ligand, DNAM-1, on T and NK cells. Finally, DSP502 inhibited tumor growth by potentiating antitumor immunity in xenograft ovarian and lung cancer models. Collectively, these findings demonstrate that DSP502, by blocking PVR and PD-L1 pathways, has dual ICI activity and holds potential therapeutic benefits for cancers such as NSCLC.