Therapeutic Combinations Research Articles

Abstract 2119: Translational research to facilitate development of novel therapeutic combinations of letrozole for the treatment of glioblastoma

Abstract Letrozole (LTZ), a third-generation aromatase inhibitor, appears to be a promising novel therapeutic for the treatment of glioblastoma (GBM). It synergistically enhances the efficacy of the DNA alkylating agent temozolomide, one of the few approved chemotherapeutics for GBM, against patient-derived GBM cells. RNA-seq analysis of tumor from recurrent GBM patients treated with LTZ prior to surgery in a “window of opportunity” phase 0/1 dose escalation clinical trial revealed that LTZ downregulates genes encoding for DNA-damage repair proteins (BRCA2) in a dose dependent manner. Our central hypothesis for this project is that combination of LTZ with targeted therapeutic agents that promote DNA damage results in synergistic effects against GBM. The objective of this study is to evaluate the effects of poly-ADP ribose polymerase (PARP) inhibitors in combination with LTZ against patient-derived GBM cells. Our secondary aim for this project is to delineate the blood-brain barrier (BBB) permeability of PARP inhibitors. Employing patient-derived G43, G75 and JHH-136 GBM lines we assessed the influence of PARP inhibitors (olaparib, pamiparib, veliparib and niraparib) in combination with LTZ on cell viability and neurosphere growth. We then utilized male CD-IGS rats (n=6; body wt., 250g) to assess plasma and brain extracellular fluid (ECF) pharmacokinetics (PK) of PARP inhibitors using cerebral microdialysis. The IC50 values for PARP inhibitors in these assays ranged from 0.06 to 11.31 µM. Treatment in combination with LTZ (0.05 µM), a non-cytotoxic concentration of LTZ in these lines, significantly decreased the IC50 values for PARP inhibitors (0.012 to 0.51 µM). Combination Index analysis indicated that this combination was strongly synergistic. Furthermore, the plasma and brain ECF PK analyses suggested that PARP inhibitors such as pamiparib and niraparib exhibit facile transport across the BBB barrier as reflected in Kp,uu,brain (ratio of AUCecf/AUCplasma, unbound) values of 0.98 and 0.45, respectively. Overall, these results provide a strong foundation for pursuing further development of combination therapy of GBM with LTZ and PARP inhibitors. A careful assessment of the clinical efficacy and safety profile of these agents will further guide shortlisting combination of LTZ for pre-clinical and clinical development. Citation Format: Aniruddha Sunil Karve, Bhavesh Babulal Gabani, Sidharth Nitin Gadgil, Shravani Parag Kulkarni, Gary A. Gudelsky, Pankaj B. Desai. Translational research to facilitate development of novel therapeutic combinations of letrozole for the treatment of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2119.

Abstract 4615: HC-7366, a potent GCN2 activator, complements belzutifan, a HIF-2⍺ antagonist, by providing combination benefit in belzutifan-sensitive models and monotherapy activity in belzutifan-resistant models

Abstract Recent approval of the HIF-2α antagonist belzutifan (Welireg) for patients with VHL disease has enabled the clinical opportunity to target the HIF-2 axis in cancer. This is particularly important for patients with clear cell renal cell carcinoma (ccRCC), in which 90% of tumors are VHL deficient, and approximately one in four primary tumors express HIF-2α exclusively. Clinical studies to date suggest an overall response rate (ORR) of 20-25% for belzutifan monotherapy, highlighting the need for a rational combination strategy to improve patient outcomes. This has led to multiple clinical trials testing belzutifan in combination with diverse therapeutic agents, including immune-checkpoint inhibitors, VEGFR-TKIs, and CDK4/6 inhibitors. HiberCell is developing a first-in-class GCN2 activator currently undergoing Ph1 clinical evaluation in patients with solid tumors (NCT05121948). GCN2 activation by HC-7366 drives the Integrated Stress Response (ISR), resulting in translation inhibition and delayed cell cycle progression in cancer cells. In preclinical studies, HC-7366 demonstrated significant antitumor activity associated with HIF suppression across diverse tumor models, including ccRCC (70% TGI), head and neck (33% regression), sarcoma (84% TGI) and prostate (100% TGI). Analysis of tumor samples revealed that HC-7366 inhibited both HIF-1α and HIF-2α, as well as multiple cell cycle regulators. Given the mechanistic link between HIF-2 and cell cycle regulation, and the noted combination benefit reported when CDK inhibitors are combined with belzutifan, we hypothesized that HC-7366 would enhance the antitumor effects of belzutifan in RCC. Combination studies in HIF-2 dependent 786-O and A-498 RCC xenografts demonstrated that HC-7366 improved the antitumor effects of belzutifan at clinically relevant doses. The combination of HC-7366 and belzutifan in A-498 drove robust tumor inhibition (~90% TGI). In 786-O, HC-7366 increased the number of complete responses from 2 of 8 tumors in the belzutifan-treated animals to 6 of 8 in the combination group. When tested in two belzutifan-resistant PDX models, HC-7366 resulted in significant monotherapy activity, achieving 17% regression as a maximal response, and inhibiting both HIF and cell cycle markers. Belzutifan is currently being tested in indications beyond RCC, so we evaluated the combination in VHLwt endometrial cancer model, MFE-280. Consistent with RCC models, the combination of HC-7366 and belzutifan resulted in tumor stasis with activation of ISR, enhanced inhibition of HIF and cell cycle markers in the combination vs belzutifan monotherapy. Together, these findings suggest that HC-7366 enhances belzutifan activity and serves as a rationale supporting clinical evaluation of this therapeutic combination. Citation Format: Michael E. Stokes, Feven Tameire, Paulina Wojnarowicz, Sho Fujisawa, Crissy Dudgeon, Sharon Huang, Nick Collette, Ben Harrison, Ashley LaCayo, Xiaohong Qiu, Takashi Kangas, Weiyu Zhang, Jeremy Drees, David Surguladze, Eric S. Lightcap, Nandita Bose. HC-7366, a potent GCN2 activator, complements belzutifan, a HIF-2⍺ antagonist, by providing combination benefit in belzutifan-sensitive models and monotherapy activity in belzutifan-resistant models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4615.

Abstract 3238: Therapeutic targeting of the genome guardian sirtuins in metastatic prostate cancer

Abstract Introduction: The effective treatment paradigm for the lethal form of metastatic prostate cancer (mPCa) remains challenging. Almost all mPCas eventually develops therapy resistance against androgen receptor (AR) inhibitors (e.g., Enzalutamide, Abiraterone) or to poly (adenosine diphosphate-ribose) polymerase (PARP) inhibitors (Olaparib, Niraparib, Talazoparib) (HRRd) when used as monotherapy. Although the patients in the ARSi+PARPi combination produced significantly longer radiologic progression-free survival (rPFS), a substantial number had attained therapy resistance by the end of the study tenure. So, a newer therapy is urgently needed. In recent years, a new targetable therapeutics development domain has been unfolding with an expanding understanding of the role of SIRTuins in prostate cancer cell survival, invasion, and progression to an aggressive metastatic state. Here, we will investigate the therapeutic efficacy of SIRT1/7 inhibitor (SIRT1/7i) and determine synergy with DNA-damaging PARP inhibitor combinations through a battery of cell and molecular assays using various prostate cancer cellular models. Methods: We have treated the mPCa cell lines 22Rv1, LAPC4, PC3 and LnCAP as well as prostate epithelial cell line RWPE1 with SIRT1 inhibitor SIRT1-IN-2 (MedChemExpress, HY-146689), SIRT7 inhibitor 97491 (MedChemExpress, HY-135899) and AR inhibitor Enzalutamide individually as well as in combination of PARP inhibitor Olaparib. The treatment of the SIRT1 and SIRT7 monotherapy arm was 72 hours long, and combination therapy continued for 7 days. The cell viability was assessed with CellTiter-Glo® 2.0 Cell Viability Assay (Promega). The cell cycle progression and proliferation analysis were done using flow cytometry through Propidium iodide staining and AnnexinV-FITC apoptosis detection chemistry. Cell survival was orthogonally assessed using colony counting assays. We adopt RAD51 foci counting assays and COMET assays for evaluating treatment-induced genome instability. Results: The preliminary result suggested that the SIRT1i+Olaparib and SIRT7i+Olaparib combination significantly sensitized the mPCa cell lines 22Rv1, PC3, and LnCAP for cell death compared to the prostate epithelial cell RWPE1. Upon the combination treatment, the mPCa cell lines showed elevated apoptotic cell death, reconfirmed through colony counting assay. Conclusion: Therapeutic combination treatment of SIRT1 and SIRT7 inhibition with Olaparib could be an effective treatment option for aggressive metastatic prostate cancer. Citation Format: Subhajit Giri. Therapeutic targeting of the genome guardian sirtuins in metastatic prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3238.

Abstract 3308: A selective FGFR1/2 degrader overcomes endocrine resistance in ER+/FGFR1-amplified andFGFR1/2-mutant breast cancer

Abstract FGFR1 amplification and FGFR1/2 activating mutations are associated with resistance to antiestrogens in the estrogen receptor-positive (ER+) breast cancer. In this study, we investigated selective degradation of FGFR1/2 using the proteolysis-targeted chimera (PROTAC) DGY-09-192 as a novel therapeutic strategy to overcome resistance to antiestrogens in ER+ breast cancers harboring FGFR1-amplification or FGFR1/2 activating mutations. Treatment of ER+/FGFR1-amplified CAMA1 and MDA-MB-134 breast cancer cells with DGY-09-192 degraded FGFR1 and suppressed phosphorylation of FGFR1 and its downstream targets FRS2, AKT, and ERK1/2. DGY-09-192-mediated FGFR1 degradation were rescued by a proteasome inhibitor, MG132, suggesting that the antitumor activity of DGY-09-192 depends on proteasome-mediated degradation. Growth of CAMA1 and MDA-MB-134 cells were modestly inhibited by single-agent DGY-09-192 or the ER degrader fulvestrant, but the combination completely arrested cell proliferation. Treatment of female NSG mice bearing established ER+/FGFR1-amplified patient-derived xenografts (PDXs) with single-agent DGY-09-192 (40 mg/kg/day, i.p.) or fulvestrant (5 mg/week, s.c.) delayed tumor growth, respectively. However, treatment with DGY-09-192 + fulvestrant induced tumor regression in all mice (n=7), suggesting that a FGFR1 degrader plus an antiestrogen can serve as an effective therapeutic combination for ER+/FGFR1-amplified breast cancer. Next, we examined whether ER+ breast cancer cells harboring FGFR1/2 hotspot mutations are sensitive to DGY-09-192. Ectopic expression of FGFR1N546K, the most common FGFR1 mutation in breast cancer, activated FGFR1 signaling and induced resistance to fulvestrant compared to MCF7 cells transduced with wild-type FGFR1. EFM-19 cells harboring FGFR2K659E exhibited resistance to fulvestrant in vitro. Treatment of both cell lines with DGY-09-192 degraded the mutant FGFR1N546K and FGFR2K659E, respectively, blocked phosphorylation of the FGFR downstream targets, and markedly inhibited cell proliferation. These results suggest that PROTAC-mediated degradation of FGFR1/2 effectively blocks the growth of ER+, FGFR1/2-mutant breast cancers resistant to antiestrogens. Conclusions: DGY-09-192 induced FGFR1/2 degradation and blocks FGFR downstream signaling in ER+/FGFR1-amplified and FGFR1/2-mutant breast cancer cells. The combination of DGY-09-192 and fulvestrant exhibited superior antitumor effects relative to each monotherapy alone in vitro and/or in vivo. Therefore, PROTAC-mediated FGFR1/2 degradation represents a promising therapeutic strategy for ER+ breast cancers harboring FGFR1-amplification or FGFR1/2 activating mutations. Citation Format: Yasuaki Uemoto, Chang-Ching Lin, Bingnan Wang, Dan Ye, Emmanuel Bikorimana, Alberto Servetto, Luigi Formisano, Fabiana Napolitano, Rosario Chica Parrado, Saurabh Mendiratta, Chuo Chen, Ariella B. Hanker, Carlos L. Arteaga. A selective FGFR1/2 degrader overcomes endocrine resistance in ER+/FGFR1-amplified andFGFR1/2-mutant breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3308.

Combined KRAS-MAPK pathway inhibitors and HER2-directed drug conjugate is efficacious in pancreatic cancer

Targeting the mitogen-activated protein kinase (MAPK) cascade in pancreatic ductal adenocarcinoma (PDAC) remains clinically unsuccessful. We aim to develop a MAPK inhibitor-based therapeutic combination with strong preclinical efficacy. Utilizing a reverse-phase protein array, we observe rapid phospho-activation of human epidermal growth factor receptor 2 (HER2) in PDAC cells upon pharmacological MAPK inhibition. Mechanistically, MAPK inhibitors lead to swift proteasomal degradation of dual-specificity phosphatase 6 (DUSP6). The carboxy terminus of HER2, containing a TEY motif also present in extracellular signal-regulated kinase 1/2 (ERK1/2), facilitates binding with DUSP6, enhancing its phosphatase activity to dephosphorylate HER2. In the presence of MAPK inhibitors, DUSP6 dissociates from the protective effect of the RING E3 ligase tripartite motif containing 21, resulting in its degradation. In PDAC patient-derived xenograft (PDX) models, combining ERK and HER inhibitors slows tumour growth and requires cytotoxic chemotherapy to achieve tumour regression. Alternatively, MAPK inhibitors with trastuzumab deruxtecan, an anti-HER2 antibody conjugated with cytotoxic chemotherapy, lead to sustained tumour regression in most tested PDXs without causing noticeable toxicity. Additionally, KRAS inhibitors also activate HER2, supporting testing the combination of KRAS inhibitors and trastuzumab deruxtecan in PDAC. This study identifies a rational and promising therapeutic combination for clinical testing in PDAC patients.

Chitosan Membranes Stabilized with Varying Acyl Lengths Release Cis-2-Decenoic Acid and Bupivacaine at Controlled Rates and Inhibit Pathogenic Biofilm.

Adherence of complex bacterial biofilm communities to burned tissue creates a challenge for treatment, with infection causing 51% of burn victim deaths. This study evaluated the release of therapeutics from wound care biomaterials and their antimicrobial activity against pathogens Staphylococcus aureus, Acinetobacter baumannii, and Pseudomonas aeruginosa. Electrospun chitosan membranes (ESCMs) were fabricated and acylated with chain lengths ranging from 6-10 carbons then loaded with 0.15 mg of anti-biofilm agent, cis-2-decenoic acid (C2DA), and 0.5 mg of local anesthetic, bupivacaine. Combinations of therapeutics released from modified ESCMs at a cumulative amount of 45-70% of bupivacaine and less than 20% of C2DA. Results from bacterial studies suggest that this combination reduced biofilm 10-fold for S. aureus, 2-fold for Acinetobacter baumannii, and 2-3-fold for Pseudomonas aeruginosa by 24 hours. Additionally, dual loaded groups reduced planktonic Staphylococcus aureus ~4-fold by 24 hours as well as Acinetobacter baumannii ~3-fold by 48 hours. The combination of therapeutics used has a significant role in biofilm prevention for selected strains via direct contact or diffusion in aqueous solutions.

Predicting efficacy assessment of combined treatment of radiotherapy and nivolumab for NSCLC patients through virtual clinical trials using QSP modeling.

Non-Small Cell Lung Cancer (NSCLC) remains one of the main causes of cancer death worldwide. In the urge of finding an effective approach to treat cancer, enormous therapeutic targets and treatment combinations are explored in clinical studies, which are not only costly, suffer from a shortage of participants, but also unable to explore all prospective therapeutic solutions. Within the evolving therapeutic landscape, the combined use of radiotherapy (RT) and checkpoint inhibitors (ICIs) emerged as a promising avenue. Exploiting the power of quantitative system pharmacology (QSP), we undertook a study to anticipate the therapeutic outcomes of these interventions, aiming to address the limitations of clinical trials. After enhancing a pre-existing QSP platform and accurately replicating clinical data outcomes, we conducted an in-depth study, examining different treatment protocols with nivolumab and RT, both as monotherapy and in combination, by assessing their efficacy through clinical endpoints, namely time to progression (TTP) and duration of response (DOR). As result, the synergy of combined protocols showcased enhanced TTP and extended DOR, suggesting dual advantages of extended response and slowed disease progression with certain combined regimens. Through the lens of QSP modeling, our findings highlight the potential to fine-tune combination therapies for NSCLC, thereby providing pivotal insights for tailoring patient-centric therapeutic interventions.

Molecular Imaging for Lung Cancer: Exploring Small Molecules, Peptides, and Beyond in Radiolabeled Diagnostics.

It is evident that radiolabeled drug delivery systems hold great promise in the field of lung cancer management. The combination of therapeutic agents with radiotracers not only allows for precise localization within lung tumors but also enables real-time monitoring of drug distribution. This approach has the potential to enhance targeted therapy and improve patient outcomes. The integration of advanced imaging modalities, such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT), has played a crucial role in the non-invasive tracking of radiolabeled drugs. These techniques provide valuable insights into drug pharmacokinetics, biodistribution, and tumor-targeting efficiency, offering clinicians the ability to personalize treatment regimens. The comprehensive analysis of preclinical and clinical studies presented in this review underscores the progress made in the field. The evidence suggests that radiolabeled drug delivery systems have the potential to revolutionize oncology by offering precise, targeted, and image-guided therapeutic interventions for lung cancer. This innovative approach not only enhances the effectiveness of treatment but also contributes to the development of personalized medicine strategies, tailoring interventions to the specific characteristics of each patient's cancer. The ongoing research in this area holds promise for further advancements in lung cancer management, potentially leading to improved outcomes and quality of life for patients.

Targeted therapies prime oncogene-driven lung cancers for macrophage-mediated destruction.

Macrophage immune checkpoint inhibitors, such as anti-CD47 antibodies, show promise in clinical trials for solid and hematologic malignancies. However, the best strategies to use these therapies remain unknown, and ongoing studies suggest they may be most effective when used in combination with other anticancer agents. Here, we developed an unbiased, high-throughput screening platform to identify drugs that render lung cancer cells more vulnerable to macrophage attack, and we found that therapeutic synergy exists between genotype-directed therapies and anti-CD47 antibodies. In validation studies, we found that the combination of genotype-directed therapies and CD47 blockade elicited robust phagocytosis and eliminated persister cells in vitro and maximized antitumor responses in vivo. Importantly, these findings broadly applied to lung cancers with various RTK/MAPK pathway alterations - including EGFR mutations, ALK fusions, or KRASG12C mutations. We observed downregulation of β2-microglobulin and CD73 as molecular mechanisms contributing to enhanced sensitivity to macrophage attack. Our findings demonstrate that dual inhibition of the RTK/MAPK pathway and the CD47/SIRPa axis is a promising immunotherapeutic strategy. Our study provides strong rationale for testing this therapeutic combination in patients with lung cancers bearing driver mutations.

Dabrafenib Alters MDSC Differentiation and Function by Activation of GCN2.

An important, but poorly understood, aspect of targeted therapeutics for cancer is the effect on antitumor immune responses. This article shows that off-target effects of dabrafenib activating the kinase GCN2 impact MDSC development and function reducing PMN-MDSCs in vitro and in vivo. This has important implications for our understanding of how this BRAF inhibitor impacts tumor growth and provides novel therapeutic target and combination possibilities.

Broad Epitope Coverage of Therapeutic Multi-Antibody Combinations Targeting SARS-CoV-2 Boosts In Vivo Protection and Neutralization Potency to Corner an Immune-Evading Virus.

Therapeutic antibodies (Abs) which act on a broader range of epitopes may provide more durable protection against the genetic drift of a target, typical of viruses or tumors. When these Abs exist concurrently on the targeted antigen, several mechanisms of action (MoAs) can be engaged, boosting therapeutic potency. This study selected combinations of four and five Abs with non- or partially overlapping epitopes to the SARS-CoV-2 spike glycoprotein, on or outside the crucial receptor binding domain (RBD), to offer resilience to emerging variants and trigger multiple MoAs. The combinations were derived from a pool of unique-sequence scFv Ab fragments retrieved from two SARS-CoV-2-naïve human phage display libraries. Following recombinant expression to full-length human IgG1 candidates, a biolayer interferometric analysis mapped epitopes to bins and confirmed that up to four Abs from across the bins can exist simultaneously on the spike glycoprotein trimer. Not all the bins of Abs interfered with the spike protein binding to angiotensin converting enzyme 2 (ACE2) in competitive binding assays, nor neutralized the pseudovirus or authentic virus in vitro, but when combined in vivo, their inclusion resulted in a much stronger viral clearance in the lungs of intranasally challenged hamsters, compared to that of those treated with mono ACE2 blockers. In addition, the Ab mixtures activated in vitro reporter cells expressing Fc-gamma receptors (FcγRs) involved in antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP). The best four-Ab combination neutralized seventeen variants of concern from Wuhan-Hu1 to Omicron BA.4/BA.5 in vitro.

The Novel ATR Inhibitor M1774 Induces Replication Protein Overexpression and Broad Synergy with DNA-targeted Anticancer Drugs.

Ataxia telangiectasia and Rad3-related (ATR) checkpoint kinase inhibitors are in clinical trials. Here we explored the molecular pharmacology and therapeutic combination strategies of the oral ATR inhibitor M1774 (Tuvusertib) with DNA-damaging agents (DDA). As single agent, M1774 suppressed cancer cell viability at nanomolar concentrations, showing greater activity than ceralasertib and berzosertib, but less potency than gartisertib and elimusertib in the small cell lung cancer H146, H82, and DMS114 cell lines. M1774 also efficiently blocked the activation of the ATR-CHK1 checkpoint pathway caused by replication stress induced by TOP1 inhibitors. Combination with non-toxic dose of M1774 enhanced TOP1 inhibitor-induced cancer cell death by enabling unscheduled replication upon replicative damage, thereby increasing genome instability. Tandem mass tag-based quantitative proteomics uncovered that M1774, in the presence of DDA, forces the expression of proteins activating replication (CDC45) and G2-M progression (PLK1 and CCNB1). In particular, the fork protection complex proteins (TIMELESS and TIPIN) were enriched. Low dose of M1774 was found highly synergistic with a broad spectrum of clinical DDAs including TOP1 inhibitors (SN-38/irinotecan, topotecan, exatecan, and exatecan), the TOP2 inhibitor etoposide, cisplatin, the RNA polymerase II inhibitor lurbinectedin, and the PARP inhibitor talazoparib in various models including cancer cell lines, patient-derived organoids, and mouse xenograft models. Furthermore, we demonstrate that M1774 reverses chemoresistance to anticancer DDAs in cancer cells lacking SLFN11 expression, suggesting that SLFN11 can be utilized for patient selection in upcoming clinical trials.

Expression of Immunotherapy Target PRAME in Cancer Correlates with Histone H3 Acetylation and Is Unrelated to Expression of Methylating (DMNT3A/3B) and Demethylating (TET1) Enzymes.

Background/Objectives: Preferentially expressed antigen in melanoma (PRAME), a member of the cancer testis antigen family, is a promising target for cancer immunotherapy. Understanding the epigenetic mechanisms involved in the regulation of PRAME expression might be crucial for optimizing anti-PRAME treatments. Methods: Three malignancies of different lineages (sinonasal melanoma, testicular seminoma, and synovial sarcoma), in which immunohistochemical (IHC) reactivity for PRAME is a common yet variable feature, were studied. The expression of PRAME, ten-eleven translocation demethylase 1 (TET1), and DNA methyltransferase (DNMT) 3A and 3B were evaluated using immunohistochemistry. Moreover, the expression of two epigenetic marks, 5-hydroxymethylcytosine (5hmC) and histone 3 acetylation (H3ac), was tested. Results: All PRAME-positive tumors expressed medium-to-high levels of H3ac but differed considerably with respect to other markers. In seminomas, PRAME expression correlated with TET1, but in melanomas and synovial sarcomas, it correlated with both DNMTs and DNMT3A, respectively. Conclusions: PRAME expression was not determined by a balance between the global expression of DNA methylating/demethylating enzymes. However, histone acetylation may be one of the epigenetic mechanisms involved in PRAME regulation. Thus, the therapeutic combination of histone deacetylase inhibitors and PRAME immunotherapy merits further investigation.

Medical Advancements in Benign Prostatic Hyperplasia Treatments.

This review aims to identify and summarize the current literature on the most recent therapeutic agents and combination strategies for the medical management of lower urinary tract symptoms resulting from benign prostatic hyperplasia. The latest advancements in BPH therapy have been in combination strategies. Alpha blockers continue to be the mainstay of treatment, but research is exploring the synergistic benefits of combining them with 5-alpha reductase inhibitors (5-ARIs), phosphodiesterase-5 (PDE5) inhibitors, and beta-3 agonists. The alpha-blocker + 5-ARI combination remains ideal for enlarged, significantly reducing clinical progression risk compared to monotherapy. Alpha-blocker + PDE5 inhibitor combinations appear safe and potentially beneficial for men with concomitant erectile dysfunction; sildenafil might hold an edge over tadalafil based on limited data. Beta-3 agonists show synergistic effects with alpha blockers for residual storage symptoms, offering similar efficacy to anticholinergics but with a better side effect profile.

Abstract IA004: Targeting clonal heterogeneity in treatment-refractory Glioblastoma with novel and empiric immunotherapies

Abstract Despite aggressive multimodal therapy, glioblastoma (GBM) remains the most common malignant primary brain tumor in adults. With the advent of therapies that revitalize the anti-tumor immune response, several immunotherapeutic modalities have been developed for the treatment of GBM. In this talk, we summarize recent clinical and pre-clinical efforts to develop immunotherapeutic and chimeric antigen receptor (CAR) T cell treatment strategies for GBM recurrence, including challenges in the form of fundamental mechanisms of therapy evasion by tumor cells, such as immense intratumoral heterogeneity, suppression of the tumor immune microenvironment, and low mutational burden. Insights from these advances and challenges have shaped our development of a translational research pipeline from initial target discovery, through target validation and exploration of mechanism, to building new biotherapeutics against novel cancer targets, and preclinical testing in our patient-derived animal models of treatment-resistant GBM. We have tracked GBM cell populations that undergo clonal evolution as a result of selective pressures exerted by standard treatments to identify the cellular composition of the recurrence, to allow us to determine the intracellular signaling pathways that drive GBM relapse. Ultimately, we will design rational combinations of therapeutics that can reignite the anti-tumor immune response, effectively and specifically target tumor cells, and reliably decrease tumor burden for GBM patients. Citation Format: Sheila K Singh. Targeting clonal heterogeneity in treatment-refractory Glioblastoma with novel and empiric immunotherapies [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr IA004.

Abstract P26: Altered RNA Export Sensitizes to Nuclear Export Inhibition in SF3B1 Mutant MDS

Abstract SF3B1 mutations, the most frequent spliceosomal alterations across cancers, occur in 25% of myelodysplastic syndromes (MDS) patients yet lack effective therapies. Two phase 2 clinical trials with the XPO1 inhibitors in high-risk MDS revealed increased activity in patients with SF3B1 mutations. XPO1 (Exportin-1) plays a role in the transport of multiple RNA species, including small nuclear RNAs (snRNAs) out of the nucleus. Given the role of XPO1 in exporting snRNAs, which form the catalytic portion of the spliceosome, we hypothesized that XPO1 inhibition may preferentially affect SF3B1-mutant cells and that SF3B1-mutant high-risk MDS patients would have a better response to rational targeted drug combinations with XPO1 inhibitors. To evaluate the mechanism underlying the preferential sensitivity of SF3B1-mutant cells to XPO1 inhibition, we conducted subcellular RNA sequencing before and after XPO1 inhibition in SF3B1 wildtype and mutant cells. Transcriptomic analysis revealed increased global retention of RNA transcripts and elevated snRNAs in the nucleus after XPO1 inhibition in the SF3B1 mutant cell line. Global RNA expression and splicing analysis revealed increased alternative splicing in SF3B1 mutant cells after XPO1 inhibition. These results suggest that the preferential sensitivity of SF3B1 mutant cells to nuclear export inhibition arises through increased nuclear retention of spliceosomal snRNAs and select mRNAs that results in perturbation of apoptotic pathways. Despite the promising efficacy of XPO1 inhibition in SF3B1-mutated MDS, dose escalation is limited by toxicity. In order to identify novel drug combination targets with lower XPO1 inhibitor doses, we employed whole genome CRISPR screens in two acute myeloid leukemia (AML) cell lines treated with XPO1 inhibitor. We identified two drug targets that greatly synergized with eltanexor specifically in the SF3B1 mutant cell lines: venetoclax (a BCL2 inhibitor), and A1331852 (a BCL-XL inhibitor). In addition, BH3 profiling demonstrated increased priming of the BCL2 and BCL-XL in SF3B1 mutant cells. We further validated these combinations in vivo using competitive transplant assays in Sf3b1 K700E conditional knock-in mice. The combination of eltanexor with venetoclax and eltanexor with A1331852 showed a significant decrease in the Sf3b1-mutant burden in the peripheral blood and bone marrow progenitor compartments, suggesting a preferential sensitivity of the combinations for SF3B1 mutant cells. Although A1331852 exhibited similar results to venetoclax, there was increased weight loss and decrease in hemoglobin associated with BCLXL inhibition. In conclusion, our study provides insight on the mechanisms underlying the heightened sensitivity of XPO1 inhibition in SF3B1-mutant MDS/AML. The identification of BCL2 and BCL-XL as synergistic targets with XPO1 inhibitors, validated by in vivo testing, BH3 profiling, and RNA sequencing, highlights the potential for therapeutic combinations. Specifically, the combination of eltanexor and venetoclax could be a promising SF3B1-specific therapy. Citation Format: Sana Chaudhry, Felipe Beckedorff, Shaista Shabbir Jasdanwala, Tulasigeri M Totiger, Maurizio Affer, Nidhi Hariramani, Olivia Tonini, Stephan Noudali, Alexandra Chirino, Alyssa Cornista, Skye Montoya, Daniel Bilbao, Jumana Afaghani, Jose Antonio Rodríguez, Shruti Bhatt, Eric Wang, Justin Taylor. Altered RNA Export Sensitizes to Nuclear Export Inhibition in SF3B1 Mutant MDS [abstract]. In: Proceedings of the Blood Cancer Discovery Symposium; 2024 Mar 4-6; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(2_Suppl):Abstract nr P26.

Assessment of wound healing activity in diabetic mice treated with a novel therapeutic combination of selenium nanoparticles and platelets rich plasma

Diabetic wound healing is sluggish, often ending in amputations. This study tested a novel, two-punch therapy in mice—Selenium nanoparticles (Se NPs) and platelet-rich plasma (PRP)—to boost healing. First, a mouse model of diabetes was created. Then, Se NPs were crafted for their impressive antioxidant and antimicrobial powers. PRP, packed with growth factors, was extracted from the mice's blood. Wound healing was tracked for 28 days through photos, scoring tools, and tissue analysis. Se NPs alone spurred healing, and PRP added extra fuel. Furthermore, when used in combination with PRP, the healing process was accelerated due to the higher concentration of growth factors in PRP. Notably, the combination of Se NPs and PRP exhibited a synergistic effect, significantly enhancing wound healing in diabetic mice. These findings hold promise for the treatment of diabetic wounds and have the potential to reduce the need for lower limb amputations associated with diabetic foot ulcers. The innovative combination therapy using Se NPs and PRP shows great potential in expediting the healing process and addressing the challenges of impaired wound healing in individuals with diabetes. This exciting finding suggests this therapy could change diabetic wound management, potentially saving limbs and improving lives.

Targeted deletion of CD244 on monocytes promotes differentiation into anti-tumorigenic macrophages and potentiates PD-L1 blockade in melanoma

BackgroundIn the myeloid compartment of the tumor microenvironment, CD244 signaling has been implicated in immunosuppressive phenotype of monocytes. However, the precise molecular mechanism and contribution of CD244 to tumor immunity in monocytes/macrophages remains elusive due to the co-existing lymphoid cells expressing CD244.MethodsTo directly assess the role of CD244 in tumor-associated macrophages, monocyte-lineage-specific CD244-deficient mice were generated using cre-lox recombination and challenged with B16F10 melanoma. The phenotype and function of tumor-infiltrating macrophages along with antigen-specific CD8 T cells were analyzed by flow cytometry and single cell RNA sequencing data analysis, and the molecular mechanism underlying anti-tumorigenic macrophage differentiation, antigen presentation, phagocytosis was investigated ex vivo. Finally, the clinical feasibility of CD244-negative monocytes as a therapeutic modality in melanoma was confirmed by adoptive transfer experiments.ResultsCD244fl/flLysMcre mice demonstrated a significant reduction in tumor volume (61% relative to that of the CD244fl/fl control group) 14 days after tumor implantation. Within tumor mass, CD244fl/flLysMcre mice also showed higher percentages of Ly6Clow macrophages, along with elevated gp100+IFN-γ+ CD8 T cells. Flow cytometry and RNA sequencing data demonstrated that ER stress resulted in increased CD244 expression on monocytes. This, in turn, impeded the generation of anti-tumorigenic Ly6Clow macrophages, phagocytosis and MHC-I antigen presentation by suppressing autophagy pathways. Combining anti-PD-L1 antibody with CD244−/− bone marrow-derived macrophages markedly improved tumor rejection compared to the anti-PD-L1 antibody alone or in combination with wild-type macrophages. Consistent with the murine data, transcriptome analysis of human melanoma tissue single-cell RNA-sequencing dataset revealed close association between CD244 and the inhibition of macrophage maturation and function. Furthermore, the presence of CD244-negative monocytes/macrophages significantly increased patient survival in primary and metastatic tumors.ConclusionOur study highlights the novel role of CD244 on monocytes/macrophages in restraining anti-tumorigenic macrophage generation and tumor antigen-specific T cell response in melanoma. Importantly, our findings suggest that CD244-deficient macrophages could potentially be used as a therapeutic agent in combination with immune checkpoint inhibitors. Furthermore, CD244 expression in monocyte-lineage cells serve as a prognostic marker in cancer patients.

Inhibition of TNBC Cell Growth by Paroxetine: Induction of Apoptosis and Blockage of Autophagy Flux.

The strategy of drug repurposing has gained traction in the field of cancer therapy as a means of discovering novel therapeutic uses for established pharmaceuticals. Paroxetine (PX), a selective serotonin reuptake inhibitor typically utilized in the treatment of depression, has demonstrated promise as an agent for combating cancer. Nevertheless, the specific functions and mechanisms by which PX operates in the context of triple-negative breast cancer (TNBC) remain ambiguous. This study aimed to examine the impact of PX on TNBC cells in vitro as both a standalone treatment and in conjunction with other pharmaceutical agents. Cell viability was measured using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, apoptosis was assessed through flow cytometry, and the effects on signaling pathways were analyzed using RNA sequencing and Western blot techniques. Furthermore, a subcutaneous tumor model was utilized to assess the in vivo efficacy of combination therapy on tumor growth. The results of our study suggest that PX may activate the Ca2+-dependent mitochondria-mediated intrinsic apoptosis pathway in TNBC by potentially influencing the PI3K/AKT/mTOR pathway as well as by inducing cytoprotective autophagy. Additionally, the combination of PX and chemotherapeutic agents demonstrated moderate inhibitory effects on 4T1 tumor growth in an in vivo model. These findings indicate that PX may exert its effects on TNBC through modulation of critical molecular pathways, offering important implications for improving chemosensitivity and identifying potential therapeutic combinations for clinical use.

Elucidating molecular mechanisms and therapeutic synergy: irreversible HER2-TKI plus T-Dxd for enhanced anti-HER2 treatment of gastric cancer

BackgroundHER2-targeted therapies have improved the outcomes of HER2-positive gastric cancer (GC), yet resistance remains a challenge. We sought to explore the effects of reversible and irreversible HER2 tyrosine kinase inhibitors (TKIs) alone or in combination with the HER2-targeting antibody drug conjugate trastuzumab deruxtecan (T-Dxd).MethodsThe effects of HER2-TKIs on HER2 and downstream signaling were evaluated via Western blotting. Proteasomal inhibitors and co-immunoprecipitation assays were performed to explore the role of proteasomal degradation in HER2 expression modulation, and immunofluorescence assays were employed to explore mechanisms of HER2 internalization. The synergistic potential of the irreversible HER2-TKI pyrotinib in combination with T-Dxd was validated using growth and viability assays in anti-HER2-positive GC cell cultures and tumor growth and immunohistochemical staining assays in a mouse xenograft model.ResultsOur study revealed that reversible HER2-TKIs elevated HER2 protein levels, whereas irreversible HER2-TKIs decreased them. Pyrotinib triggered HER2 degradation within the proteasome by promoting ubiquitination and dissociation from HSP90. Furthermore, pyrotinib substantially induced HER2 internalization, which led to improved cellular uptake of T-Dxd. The increased T-Dxd uptake was accompanied by greater efficacy in suppressing the growth of GC cells and enhanced anti-tumor effects in an animal model.ConclusionIn summary, our research reveals the molecular mechanisms of irreversible HER2-TKIs in regulating HER2 protein expression by promoting HER2 internalization. These findings advance our comprehension of targeted therapy for GC and provide a promising therapeutic combination strategy with enhanced efficacy against HER2-positive GC.