Therapeutic Potential Of Combination Research Articles

Targeting tumor-associated macrophages to reverse antitumor drug resistance.

Currently, antitumor drugs show limited clinical outcomes, mainly due to adaptive resistance. Clinical evidence has highlighted the importance of the tumor microenvironment (TME) and tumor-associated macrophages (TAMs) in tumor response to conventional antitumor drugs. Preclinical studies show that TAMs following antitumor agent can be reprogrammed to an immunosuppressive phenotype and proangiogenic activities through different mechanisms, mediating drug resistance and poor prognosis. Potential extrinsic inhibitors targeting TAMs repolarize to an M1-like phenotype or downregulate proangiogenic function, enhancing therapeutic efficacy of anti-tumor therapy. Moreover, pharmacological modulation of macrophages that restore the immune stimulatory characteristics is useful to reshaping the tumor microenvironment, thus further limiting tumor growth. This review aims to introduce macrophage response in tumor therapy and provide a potential therapeutic combination strategy of TAM-targeting immunomodulation with conventional antitumor drugs.

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.

The Rac1-inhibitor EHop-016 attenuates AML cell migration and enhances the efficacy of daunorubicin in MOLM-13 transplanted zebrafish larvae

Ras-related C3 botulinum toxin substrate 1 (Rac1) is a GTPase implicated in cell migration and homing of hematopoietic cells to the hematopoietic niche, and is commonly overexpressed in acute myeloid leukemia (AML). This can lead to quiescence of leukemic blasts in the niche and reduced response to therapy. We investigated the Rac1 inhibitor EHop-016 on AML by assessing its effects on MOLM-13 cells in vitro and in zebrafish larvae, regarding cell motility and therapeutic potential in combination with daunorubicin (DNR). In vitro assessment of proliferation and viability was by measurement of 3H-thymidine incorporation and detection of Annexin V/PI positive cells. Cell motility was evaluated by measurement of migration in a transwell system. Fluorescently stained MOLM-13 cells were injected into zebrafish larvae, and individual cells followed by confocal microscopy. Cell accumulation in the caudal hematopoietic tissue (CHT) was studied using a 12-hour timelapse, while in vivo efficacy of DNR, EHop-016 or a combination was investigated over 24 h.The in vitro results showed that EHop-016 acted synergistically in combination with DNR in reducing the viability of MOLM-13 cells (Bliss synergy score above 10 %). Non-toxic concentrations of EHop-016 reduced cell migration. These findings were reproduced in zebrafish larvae: larvae receiving both DNR and EHop-016 had significantly reduced tumor burden compared to the untreated control or single treatments. The accumulation of MOLM-13 cells in the CHT was reduced in larvae receiving EHop-016 treatment.Our findings demonstrate that targeting Rac1 in AML holds promise as a complementary treatment to established chemotherapy and should be further investigated.

Unveiling immune checkpoint regulation: exploring the power of in vivo CRISPR screenings in cancer immunotherapy.

Immune checkpoint inhibitors (ICIs) have revolutionized cancer immunotherapy by reinvigorating antitumor immune responses, but their efficacy remains limited in most patients. To address this challenge and optimize Immune check inhibitor treatment, understanding the underlying molecular intricacies involved is crucial. The emergence of CRISPR-Cas9 technology has empowered researchers to precisely investigate gene function and has introduced transformative shifts in identifying key genes for various physiological and pathological processes. CRISPR screenings, particularly in vivo CRISPR screenings, have become invaluable tools in deciphering molecular networks and signaling pathways governing suppressive immune checkpoint molecules. In this review, we provide a comprehensive overview of in vivo CRISPR screenings in cancer immunotherapy, exploring how this cutting-edge technology has unraveled potential novel therapeutic targets and combination strategies. We delve into the latest findings and advancements, shedding light on immune checkpoint regulation and offering exciting prospects for the development of innovative and effective treatments for cancer patients.

Abstract B171: BX-795 enhances the efficacy of crizotinib in colorectal cancer cells by altering the activity of aurora kinases

Abstract Background: Metastatic Colorectal cancer (mCRC) is driven by various genetic mutations that, in turn, present potentially actionable targets. Recent improvements in combining therapies have shown modest benefits in patients with BRAF mutated mCRC. Several efforts focusing on various KRAS inhibitors are ongoing in mCRC with outcomes pending. With no significant advances to improve outcomes in most patients with mCRC, it is important to identify novel therapies that have the potential to significantly improve the outcomes of these patients. Aims: To perform high-throughput screening (HTS) using clinically relevant drugs to identify novel and effective therapeutic combinations for patients with KRAS/BRAF mutated mCRC. Methods: We performed unbiased HTS with ~250 drugs that were either currently in clinical trials or approved by the FDA. 57 drugs that decreased cell proliferation in both 2D and 3D cultures and were effective in both HCT116 (KRAS mutated) and HT29 (BRAF mutated) cell lines, were termed as “pan-active” drugs. These drugs were divided into 9 classes based on targets they inhibit. Using the BLISS model, synergistic drug combinations were identified by HTS where the top three effective drugs from each class of “pan-active” drugs were paired with the top three drugs of all other classes. Effects of one such combination (BX-795 with crizotinib) were further validated in vitro by cell growth assays and measuring changes in apoptotic mediators by western blotting (WB) and flow cytometry (FACS). Mechanisms of synergy were examined by Reverse Phase Protein Array (RPPA) followed by WB and immunostaining of CRC cells to assess inhibition of downstream targets. Results: Pairwise HTS studies identified the PDK1/TBK1 inhibitor BX-795 to be synergistic with the MET/ALK inhibitor crizotinib in multiple CRC cell lines. This combination was further validated in vitro by MTT and colony formation assays in various KRAS and BRAF mutated CRC cell lines. Analyses of markers for apoptosis by WB and FACS using FITC-Annexin V and PI confirmed that the drug combination increased cell death in multiple CRC cell lines as compared to single agents. Unbiased RPPA indicated that the combination led to very strong and unexpected inhibition of the Aurora kinases along with decreases in the expected AKT/mTOR pathway in multiple CRC cell lines when compared to monotherapies. Immunostaining revealed significant mitotic defects in CRC cells treated with the drug combination but not with single agents suggesting that mitotic defects was a possible mechanism of cell death. Further, combining an Aurora kinase A inhibitor with crizotinib enhanced the efficacy of crizotinib validating the proposed mechanism. Conclusions: Our unbiased HTS and validation studies demonstrated that combining BX-795 with crizotinib is a potential novel effective therapeutic combination for patients with mCRC. Our in vitro studies serve as the basis for future preclinical and, if warranted, clinical studies to determine the efficacy of this drug combination in patients with mCRC. Citation Format: Susmita Ghosh, Fan Fan, Reid Powell, Yong Sung Park, Clifford Stephan, Jason Roszik, Lee M Ellis, Rajat Bhattacharya. BX-795 enhances the efficacy of crizotinib in colorectal cancer cells by altering the activity of aurora kinases [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B171.

Microglia-neuron-vascular interactions in ischemia.

Cerebral ischemia is a devastating condition that results in impaired blood flow in the brain leading to acute brain injury. As the most common form of stroke, occlusion of cerebral arteries leads to a characteristic sequence of pathophysiological changes in the brain tissue. The mechanisms involved, and comorbidities that determine outcome after an ischemic event appearto be highly heterogeneous. On their own, the processes leading to neuronal injury in the absence of sufficient blood supply to meet the metabolic demand of the cells are complex and manifest at different temporal and spatial scales. While the contribution of non-neuronal cells to stroke pathophysiology is increasingly recognized, recent data show that microglia, the main immune cells of the central nervous system parenchyma, play previously unrecognized roles in basic physiological processes beyond their inflammatory functions, which markedly change during ischemic conditions. In this review, we aim to discuss some of the known microglia-neuron-vascular interactions assumed to contribute to the acute and delayed pathologies after cerebral ischemia. Because the mechanisms of neuronal injury have been extensively discussed in several excellent previous reviews, here we focus on some recently explored pathways that may directly or indirectly shape neuronal injury through microglia-related actions. These discoveries suggest that modulating gliovascular processes in different forms of stroke and other neurological disorders might have presently unexplored therapeutic potential in combination with neuroprotective and flow restoration strategies.

VRK3 depletion induces cell cycle arrest and metabolic reprogramming of pontine diffuse midline glioma - H3K27 altered cells.

We previously identified VRK3 as a specific vulnerability in DMG-H3K27M cells in a synthetic lethality screen targeting the whole kinome. The aim of the present study was to elucidate the mechanisms by which VRK3 depletion impact DMG-H3K27M cell fitness. Gene expression studies after VRK3 knockdown emphasized the inhibition of genes involved in G1/S transition of the cell cycle resulting in growth arrest in G1. Additionally, a massive modulation of genes involved in chromosome segregation was observed, concomitantly with a reduction in the level of phosphorylation of serine 10 and serine 28 of histone H3 supporting the regulation of chromatin condensation during cell division. This last effect could be partly due to a concomitant decrease of the chromatin kinase VRK1 in DMG following VRK3 knockdown. Furthermore, a metabolic switch specific to VRK3 function was observed towards increased oxidative phosphorylation without change in mitochondria content, that we hypothesized would represent a cell rescue mechanism. This study further explored the vulnerability of DMG-H3K27M cells to VRK3 depletion suggesting potential therapeutic combinations, e.g. with the mitochondrial ClpP protease activator ONC201.

Hypoxia-induced TMTC3 expression in esophageal squamous cell carcinoma potentiates tumor angiogenesis through Rho GTPase/STAT3/VEGFA pathway

BackgroundHypoxia is one of most typical features in the tumor microenvironment of solid tumor and an inducer of endoplasmic reticulum (ER) stress, and HIF-1α functions as a key transcription factor regulator to promote tumor angiogenesis in the adaptive response to hypoxia. Increasing evidence has suggested that hypoxia plays an important regulatory role of ER homeostasis. We previously identified TMTC3 as an ER stress mediator under nutrient-deficiency condition in esophageal squamous cell carcinoma (ESCC), but the molecular mechanism in hypoxia is still unclear.MethodsRNA sequencing data of TMTC3 knockdown cells and TCGA database were analyzed to determine the association of TMTC3 and hypoxia. Moreover, ChIP assay and dual-luciferase reporter assay were performed to detect the interaction of HIF-1α and TMTC3 promoter. In vitro and in vivo assays were used to investigate the function of TMTC3 in tumor angiogenesis. The molecular mechanism was determined using co-immunoprecipitation assays, immunofluorescence assays and western blot. The TMTC3 inhibitor was identified by high-throughput screening of FDA-approved drugs. The combination of TMTC3 inhibitor and cisplatin was conducted to confirm the efficiency in vitro and in vivo.ResultsThe expression of TMTC3 was remarkably increased under hypoxia and regulated by HIF-1α. Knockdown of TMTC3 inhibited the capability of tumor angiogenesis and ROS production in ESCC. Mechanistically, TMTC3 promoted the production of GTP through interacting with IMPDH2 Bateman domain. The activity of Rho GTPase/STAT3, regulated by cellular GTP levels, decreased in TMTC3 knockdown cells, whereas reversed by IMPDH2 overexpression. Additionally, TMTC3 regulated the expression of VEGFA through Rho GTPase/STAT3 pathway. Allopurinol inhibited the expression of TMTC3 and further reduced the phosphorylation and activation of STAT3 signaling pathway in a dose-dependent manner in ESCC. Additionally, the combination of allopurinol and cisplatin significantly inhibited the cell viability in vitro and tumor growth in vivo, comparing with single drug treatment, respectively.ConclusionsCollectively, our study clarified the molecular mechanism of TMTC3 in regulating tumor angiogenesis and highlighted the potential therapeutic combination of TMTC3 inhibitor and cisplatin, which proposed a promising strategy for the treatment of ESCC.

Abstract 1581: KSQ-4279, a first-in-class USP1 inhibitor shows strong combination activity with multiple PARP inhibitors in BRCA mutant cancers

Abstract Tumors harboring BRCA1/2 mutations and other homologous recombination deficiencies are sensitive to agents targeting pathways involved in DNA repair including poly (ADP-ribose) polymerase (PARP) inhibitors, which have been approved for the treatment of BRCA mutant cancers. Despite the clinical benefit with these drugs, many patients achieve incomplete disease control and often develop resistance. By employing our CRISPRomics® technology to screen over 600 cancer cell lines, we identified the deubiquitinating enzyme USP1 as one of the top targets that displays selective anti-tumor activity in ovarian and triple negative breast cancers with homologous recombination deficiencies. Subsequent drug discovery efforts identified KSQ-4279 as a potent and highly selective first-in-class small molecule USP1 inhibitor that is now in clinical development. In preclinical models, we previously demonstrated that KSQ-4279 shows therapeutic potential in combination with olaparib for treating patients who are either intrinsically resistant, or have developed acquired resistance to PARP inhibitors. We performed studies to investigate the therapeutic potential of combining KSQ-4279 across a variety of PARP inhibitors with different ‘PARP trapping’ potencies. Clonogenic assays demonstrated synergistic effects of KSQ-4279 across multiple PARP inhibitors regardless of their PARP trapping potency. Second generation PARP1 selective inhibitors such as AZD5305 are currently in clinical development and may provide a safety and efficacy advantage for patients. We have investigated the potential of combining KSQ-4279 with AZD5305 in a PARP refractory patient-derived triple negative breast cancer (TNBC) xenograft model by evaluating tolerability and anti-tumor efficacy. We observed significantly greater and more durable anti-tumor activity, including regressions, with the combination therapy compared to single agents. Our data supports the clinical testing of KSQ-4279 in combination with PARP inhibitors. Citation Format: Erica Tobin, Pamela Sullivan, Morgan Murray, Hugh Gannon, Anne Dodson, Sol Shenker, Frank Stegmeier, Andrew Wylie, Louise Cadzow. KSQ-4279, a first-in-class USP1 inhibitor shows strong combination activity with multiple PARP inhibitors in BRCA mutant cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1581.

Abstract 542: Synthetic lethal targeting of AURKA in head and neck squamous cell carcinoma

Abstract The purpose of this study was to identify new combination strategies for improving treatment of head and neck squamous cell carcinomas (HNSCCs). HNSCCs often express high levels of Aurora kinase A (AURKA), most studied as a kinase important for mitotic progression in normal cells. Notably the overexpression of AURKA in many cancers allows it to act throughout the cell cycle, acquiring new cancer-promoting activities. While several AURKA inhibitors are in clinical trials, limited efficacy is observed with single treatment, causing us to evaluate potential AURKA combination therapeutic combinations. Particularly relevant to HNSCC, which often overexpress and depend on EGFR, overexpression of AURKA can promote resistance to EGFR inhibitors by mechanisms variously identified as including activation of the EGFR effectors AKT, ERK, and BIM. Using viability and clonogenic assays, we have found that the AURKA inhibitor VIC-1911 synergizes with the EGFR inhibitors afatinib and erlotinib in multiple HNSCC cell lines and retains activity in HNSCC cell models selected for resistance to these EGFR inhibitors. However, AURKA inhibition increases levels of AURKA protein and its protein-stabilizing partner TPX2, counteracting drug effect. We also combined inhibition of AURKA with inhibition of WEE1, a regulator of the G2/M checkpoint, finding this dual inhibition enhanced levels of apoptosis in EGFR inhibitor resistant HNSCC cell lines. Intriguingly, one recent study reported AURKA can also modulate the glycolytic signaling pathway, increasing the cancer-promoting Warburg effect. We have found that inhibition of AURKA can significantly reduce glycolytic flux in HNSCC cell lines and alter expression and phosphorylation of key glycolytic enzymes. Dual inhibition of AURKA in combination with inhibitors of glycolytic enzymes, or enzymes regulating oxidative phosphorylation, showed marked synergy. These and other results investigating AURKA signaling mechanisms in HNSCC demonstrate that the non-canonical functions of AURKA include viable therapeutic targets that can enhance the efficacy of AURKA inhibition in HNSCC. Citation Format: Flaviane N da Silva, Theodore Nguyen, Jong Woo Lee, Barbara Burtness, Erica Golemis. Synthetic lethal targeting of AURKA in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 542.

Radiation-induced immune response in novel radiotherapy approaches FLASH and spatially fractionated radiotherapies.

The last several years have revealed increasing evidence of the immunomodulatory role of radiation therapy. Radiotherapy reshapes the tumoral microenvironment can shift the balance toward a more immunostimulatory or immunosuppressive microenvironment. The immune response to radiation therapy appears to depend on the irradiation configuration (dose, particle, fractionation) and delivery modes (dose rate, spatial distributions). Although an optimal irradiation configuration (dose, temporal fractionation, spatial dose distribution, etc.) has not yet been determined, temporal schemes employing high doses per fraction appear to favor radiation-induced immune response through immunogenic cell death. Through the release of damage-associated molecular patterns and the sensing of double-stranded DNA and RNA breaks, immunogenic cell death activates the innate and adaptive immune response, leading to tumor infiltration by effector T cells and the abscopal effect. Novel radiotherapy approaches such as FLASH and spatially fractionated radiotherapies (SFRT) strongly modulate the dose delivery method. FLASH-RT and SFRT have the potential to trigger the immune system effectively while preserving healthy surrounding tissues. This manuscript reviews the current state of knowledge on the immunomodulation effects of these two new radiotherapy techniques in the tumor, healthy immune cells and non-targeted regions, as well as their therapeutic potential in combination with immunotherapy.

Combining Metformin and Drug-Loaded Kidney-Targeting Micelles for Polycystic Kidney Disease.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease that leads to eventual renal failure. Metformin (MET), an AMP-activated protein kinase (AMPK) activator already approved for type 2 diabetes, is currently investigated for ADPKD treatment. However, despite high tolerability, MET showed varying therapeutic efficacy in preclinical ADPKD studies. Thus, newer strategies have combined MET with other ADPKD small molecule drug candidates, thereby targeting multiple ADPKD-associated signaling pathways to enhance therapeutic outcomes through potential drug synergy. Unfortunately, the off-target side effects caused by these additional drug candidates pose a major hurdle. To address this, our group has previously developed kidney-targeting peptide amphiphile micelles (KMs), which displayed significant kidney accumulation in vivo, for delivering drugs to the site of the disease. To mitigate the adverse effects of ADPKD drugs and evaluate their therapeutic potential in combination with MET, herein, we loaded KMs with ADPKD drug candidates including salsalate, octreotide, bardoxolone methyl, rapamycin, tolvaptan, and pioglitazone, and tested their in vitro therapeutic efficacy when combined with free MET. Specifically, after determining the 40% inhibitory concentration for each drug (IC40), the size, morphology, and surface charge of drug-loaded KMs were characterized. Next, drug-loaded KMs were applied in combination with MET to treat renal proximal tubule cells derived from Pkd1flox/-:TSLargeT mice in 2D proliferation and 3D cyst model. MET combined with all drug-loaded KMs demonstrated significantly enhanced efficacy as compared to free drugs in inhibiting cell proliferation and cyst growth. Notably, synergistic effects were found for MET and KMs loaded with either salsalate or rapamycin as determined by Bliss synergy scores. Together, we show drug synergy using drug-loaded nanoparticles and free MET for the first time and present a novel nanomedicine-based combinatorial therapeutic approach for ADPKD with enhanced efficacy. The online version contains supplementary material available at 10.1007/s12195-022-00753-9.

CHD4 Loss Alters Chromatin Accessibility Giving Rise to Sensitivity to DNA Damage Repair Compounds in Acute Myeloid Leukaemia

Background: Acute Myeloid Leukaemia (AML) is a heterogeneous group of blood and bone marrow disorders arising from myeloid precursors. Responses to current therapeutic options remain sub-optimal for many individuals, highlighting the need for novel therapeutic options; alongside stratifying patients by (epi)genetic alterations. Chromatin remodelling complexes (e.g. nucleosome remodelling and deacetylation complex (NuRD)), that control chromatin organization and gene expression, have emerged as key players in cancer. Cancer genome studies highlighted that modifications in NuRD occur in > 20% of cancers. NuRD regulates chromatin accessibility; CHD4, a core subunit, has a multitude of functions including epigenetic regulation, DNA Damage Repair (DDR), replication and cell cycle progression. Aims: To explore the molecular and epigenetic functions of CHD4 in AML using CRISPR-Cas9 isogenic knockout models (CHD4-/-), particularly the role of CHD4 on AML development and progression and evaluate if there is targetable therapeutic potential. Methods: TGCA datasets were assessed to identify impact of CHD4 expression on AML patient prognosis. Isogenic CHD4-/- models were generated using CRISPR-Cas9 in two AML cell lines (OCI AML 2 and OCI AML 3). Knockout models underwent functional phenotyping studies before inducing DNA damage at clinically relevant doses. Epigenetic mechanisms of CHD4 were assessed using multi-omic approaches including ATAC sequencing, RNA sequencing and proteomic analysis. A DNA Damage/Repair Compound Library (Selleckchem) was screened on CHD4-/- and CHD4 WT models using CellTox™ Green to identify DNA damage compounds that showed significant sensitivity in CHD-/- models and validation was assessed using CellTiter® Glo viability assay and co-culture assays. Combination indices (CI) were calculated using CompuSyn software. Results: Analysis of TCGA AML transcriptomic data (N=173), found that AML patients with intermediate risk cytogenetics and low CHD4 expression had a poorer 5-year survival compared to similar risk group patients with higher CHD4 levels (P=0.032). This indicates that lower CHD4 levels may result in a link between altered cellular transcription and patient survival causing a more aggressive disease phenotype in these patients. Phenotypical analysis of CHD4-/- models showed increased 53BP1 (P= 0.026) and PARP indicating DDR defect characterised by unrepaired dsDNA breaks. Defective DDR was shown by reduced proliferation with cell cycle analysis showing inter-S phase defect with accumulation in G1 and reduction in cells entering S and G2/M phases in the CHD4-/- models. With this profile being further exacerbated under cell exposure to clinically relevant irradiation induced stress. Chromatin accessibility was assessed via ATAC sequencing and showed enhanced open chromatin at areas impacting immune regulation within CHD4-/- cells. RNA and proteomic analysis both showed upregulation of genes involved in immune activation (P=6.96e-8) and cellular senescence (P=5.6e-5) pathways suggesting an altered immune profile in CHD4-/- cells with a potential to exploit this therapeutically. To identify potential therapeutic vulnerabilities in CHD4-/- cells we performed high throughput screening using a 160 compound DNA damaging library which showed an increased sensitivity of CHD4-/- cells to compounds targeting DNA/RNA synthesis pathways, including Mitomycin C, this indicated that loss of CHD4 results in hyper-increase in damage. Further investigation with Mitomycin C found that when in combination with Cytarabine yielded enhanced synergistic effects in CHD4-/- cells (CI 0.001-0.37) when compared to CHD4 WT (CI 0.002-3.1). There was also significantly less viable cells (P=0.016) 24-hours post combination treatment in CHD4-/- compared to WT (33% vs 56%), indicating that CHD4 loss is furthering the damage caused by improper cell cycle leading to cell death. Conclusion A cohort of AML patients with intermediate risk cytogenetics along with lower CHD4 levels have poorer 5-year survival. Lack of CHD4 is shown to cause epigenetic restructuring, gene abnormalities and defective DDR. We have identified Mitomycin C as a therapeutic potential in combination with Cytarabine in CHD4-/- models. Further investigation of this in combination and the mechanism of action could lead to improved outcome for this sub-group of patients.

Novel Sirpα-Fc Fusion Protein IMM01 Exhibits Dual Anti-Tumor Activities By Targeting CD47/Sirpα Signal Pathway

Background: This study aims to evaluate the anti-tumor mechanism of IMM01, a novel recombinant SIRPα-Fc fusion protein targeting CD47-SIRPα pathway. Methods: IMM01 was a recombinant fusion protein of SIRPα (V2) extracellular segment domain 1 and human IgG1 Fc, with the N glycosylation site N80 mutated to A (Alanine) in D1 region. IMM01 fusion proteins were produced using in house developed CHO-K1 cell expression system. IMM01 blocking activities on Jurkat-CSR (Jurkat-CD47KO-CARSIRPα) cells, antibody-dependent cellular phagocytosis (ADCP), antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) assays were done by flow cytometry. Detection of cytokines of IL-1β, IL-2, IL-4, IL-5, IL-6, GM-CSF, IFN-γ, IL-10 and TNF production by PBMC was done CBA assay. In vivo mechanism of action study was done in different mouse models including HL-60 CB17-SCID, Daudi CB17-SCID, SNU-1 CB17-SCID, Raji CB17-SCID and CT26-hPDL1(Tg) hCD47(Tg) mPDL1(KO) mCD47(KO). In addition, human red blood cells binding and erythrocyte agglutination of IMM01 were also evaluated. Results:In vitro binding assay revealed that IMM01 has a strong binding affinity to CD47 with lowest EC50 at 0.4967 nM. The removal of N-glycosylation modification improves the consistence and purity of the IMM01 protein expression, and the potential mitigation of immunogenicity. Using a chimeric SIRPα receptor protein expressing cell line, IMM01 not only can block the CD47 at the protein level, but also block the signal transduction at the cellular levels. Compared to negative (hIgG1-Fc) controls, IMM01 stimulated secretion of minimal amount of IL-10 and TNF but inhibited IL-8 production by PBMC. IMM01 can induce strong ADCP and moderate ADCC, but not the CDC activities. The strong phagocytosis against tumor cells is dependent on interaction of Fc with activating FcγRs on the presence of macrophages. Mechanism of action studies demonstrated that IMM01 induced strong phagocytosis against tumor cells was attributed to dual activities of blocking "don't eat me" signal and activating "eat me" signal. IMM01 has strong and robust in vivo anti-tumor activities in different mouse tumor models either as monotherapy on hematological malignancies, or in combination therapy with PD-L1 mAb, PD-1 mAb, CD33 mAb, and HER-2 mAb, respectively. The in vivo therapeutic efficacy of IMM01 was done in HL-60 xenograft model showed that 100% of the mice achieved CR after administration of IMM01 at 5mg/kg for 2 weeks. Also, 100% of mice achieved CR after IMM01 combined with the above mentioned mAbs in different mouse models, respectively. This illustrated that IMM01 has a great therapeutic potential in combination with these commonly used immunotherapy or targeted therapy agents to be used in the treatment of solid tumors and hematological malignancies. Finally, IMM01 demonstrated a favorable safety profile with no human RBC binding, nor hemagglutination induction. Conclusion: Our study results demonstrated that, as a novel bi-functional SIRPα-Fc recombinant protein, IMM01 has very strong anti-tumor activities with a good curative effect on mouse xenograft tumor models with dual anti-tumor activities by blocking CD47 "don't eat me" signal and activating the phagocytosis "eat me" signal. Furthermore, IMM01 demonstrated a well synergistic effect when combined with different immunotherapeutic agents. Importantly, our results revealed that IMM01 has a favorable safety profile with no human RBC binding activity and no hemagglutination induction. Phase I clinical study is ongoing, and the clinical trial results will be reported soon. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

EXTH-76. PRMT5 INHIBITION SENSITIZES GLIOBLASTOMA NEUROSPHERES TO TEMOZOLOMIDE

Abstract INTRODUCTION The median survival of Glioblastoma (GBM) patients is less than two years with the standard of care of maximal surgical resection, radiation, and temozolomide (TMZ) chemotherapy. Protein Arginine Methyltransferase 5 (PRMT5), which regulates cellular functions by symmetrically di-methylating arginine residues, is overexpressed in GBM. Inhibiting PRMT5 induces apoptosis in differentiated and senescence in stem-like GBM tumor cells. We inhibited PRMT5 in GBM neurospheres to determine if PRMT5 inhibition would enhance TMZ’s antitumor effect. METHODS We depleted PRMT5 activity, in vitro, using target-specific siRNA or LLY-283 and combined these with TMZ treatment. We evaluated the antitumor effect of this combination using cell viability assay, cell cycle analysis, apoptosis assay, and western blot. RESULTS TMZ reduced the viability of GBMNS with PRMT5 knockdown significantly more than the viability of PRMT5 intact GBMNS. The combination of TMZ and PRMT5 knockdown elevated the expression of cleaved caspase 3 and caspase3/7 indicating that PRMT5 knockdown enhanced the apoptotic effects of TMZ. Cell cycle analysis showed that depleting PRMT5 abrogated TMZ-induced G2/M cell cycle arrest. Further, treatment of PRMT5-depleted GBMNS with TMZ increased ɣ-H2AX expression compared PRMT5 intact GBMNS treated with TMZ, suggesting that PRMT5 depletion enhanced TMZ-induced DNA damage. PRMT5 knockdown also inhibited the symmetric di-methylation of RUVBL1 that is required for homologous recombination repair of TMZ treatment-related DNA damage. CONCLUSION Overall, PRMT5 inhibition sensitized GBMNS to TMZ and enhanced TMZ-related DNA damage and cytotoxicity. These findings support further development of this potential therapeutic combination.

ENHANCED ANTICANCER POTENTIAL OF IMIPRAMINE IN CONJUGATION WITH NIACIN IN DENA INDUCED HEPATOCELLULAR CARCINOMA

Hepatocellular carcinoma (HCC) is constantly seeking the interest of researchers as an untreatable and fatal disease, attaining second position so far worldwide. Our study provides us an opportunity to explore new potential therapeutic combinations in this regard. We hypothesized that the anticancer activity of imipramine, which is used as an antidepressant drug, can be potentiated with an adjuvant, niacin, thereby providing a better treatment approach. The combination turned out to show beneficial alterations in various liver function test, caspase 3 (antiapoptotic) activity, tumor growth and survival profile of animals, precisely in our therapeutic group involving imipramine and niacin combination post diethyl nitrosamine (DENA) dose and hepatocellular carcinoma induction. Results demonstrated a remarkable restoration in all liver enzymes in the therapeutic groups.

Repurposing the repurposed: thalidomide analogs as immune stimulants to overcome T cell exhaustion

In this issue of Cell Chemical Biology, Geng and colleagues employ a novel mouse model of humanized cereblon (Crbn) to provide insights into the immunomodulatory effects of lenalidomide and provide rationale for potential therapeutic combinations including anti-PD1 immunotherapy.

Combination Approaches to Target PD-1 Signaling in Cancer.

Cancer remains the second leading cause of death in the US, accounting for 25% of all deaths nationwide. Immunotherapy techniques bolster the immune cells’ ability to target malignant cancer cells and have brought immense improvements in the field of cancer treatments. One important inhibitory protein in T cells, programmed cell death protein 1 (PD-1), has become an invaluable target for cancer immunotherapy. While anti-PD-1 antibody therapy is extremely successful in some patients, in others it fails or even causes further complications, including cancer hyper-progression and immune-related adverse events. Along with countless translational studies of the PD-1 signaling pathway, there are currently close to 5,000 clinical trials for antibodies against PD-1 and its ligand, PD-L1, around 80% of which investigate combinations with other therapies. Nevertheless, more work is needed to better understand the PD-1 signaling pathway and to facilitate new and improved evidence-based combination strategies. In this work, we consolidate recent discoveries of PD-1 signaling mediators and their therapeutic potential in combination with anti-PD-1/PD-L1 agents. We focus on the phosphatases SHP2 and PTPN2; the kinases ITK, VRK2, GSK-3, and CDK4/6; and the signaling adaptor protein PAG. We discuss their biology both in cancer cells and T cells, with a focus on their role in relation to PD-1 to determine their potential in therapeutic combinations. The literature discussed here was obtained from a search of the published literature and ClinicalTrials.gov with the following key terms: checkpoint inhibition, cancer immunotherapy, PD-1, PD-L1, SHP2, PTPN2, ITK, VRK2, CDK4/6, GSK-3, and PAG. Together, we find that all of these proteins are logical and promising targets for combination therapy, and that with a deeper mechanistic understanding they have potential to improve the response rate and decrease adverse events when thoughtfully used in combination with checkpoint inhibitors.

Ras-Related Protein Rab-32 and Thrombospondin 1 Confer Resistance to the EGFR Tyrosine Kinase Inhibitor Osimertinib by Activating Focal Adhesion Kinase in Non-Small Cell Lung Cancer.

Simple SummaryOsimertinib is a third-generation EGFR tyrosine kinase inhibitor and the standard of care therapy for non-small cell lung cancer patients harboring EGFR-activating mutations. However, even for patients treated with osimertinib, resistance inevitably occurs leading to disease progression. Here, we utilized two osimertinib-resistant cell lines and investigated their RNA profiles. We found that Ras-related protein Rab-32 (RAB32) and thrombospondin 1 (THBS1) were upregulated and associated with resistance in osimertinib-resistant cells as well as in liquid biopsies from patients with disease progression following osimertinib treatment. Moreover, we found RAB32 and THBS1 to be mechanistically linked to activation of the focal adhesion pathway where combination of osimertinib with a FAK inhibitor resulted in a synergistic suppression of viability of osimertinib-resistant cells. Our findings propose a potential therapeutic strategy for overcoming acquired resistance to osimertinib in non-small cell lung cancer.Treatment with the tyrosine kinase inhibitor (TKI) osimertinib is the standard of care for non-small cell lung cancer (NSCLC) patients with activating mutations in the epidermal growth factor receptor (EGFR). Osimertinib is also used in T790M-positive NSCLC that may occur de novo or be acquired following first-line treatment with other EGFR TKIs (i.e., gefitinib, erlotinib, afatinib, or dacomitinib). However, patients treated with osimertinib have a high risk of developing resistance to the treatment. A substantial fraction of the mechanisms for resistance is unknown and may involve RNA and/or protein alterations. In this study, we investigated the full transcriptome of parental and osimertinib-resistant cell lines, revealing 131 differentially expressed genes. Knockdown screening of the genes upregulated in resistant cell lines uncovered eight genes to partly confer resistance to osimertinib. Among them, we detected the expression of Ras-related protein Rab-32 (RAB32) and thrombospondin 1 (THBS1) in plasmas sampled at baseline and at disease progression from EGFR-positive NSCLC patients treated with osimertinib. Both genes were upregulated in progression samples. Moreover, we found that knockdown of RAB32 and THBS1 reduced the expression of phosphorylated focal adhesion kinase (FAK). Combination of osimertinib with a FAK inhibitor resulted in synergistic toxicity in osimertinib-resistant cells, suggesting a potential therapeutic drug combination for overcoming resistance to osimertinib in NSCLC patients.

Abstract 1390: Immunotherapy boosts B cell responses in lung adenocarcinoma

Abstract Introduction: B cells constitute a major part of the lung tumour microenvironment where they are organised in tertiary lymphoid structures (TLS) that resemble antibody-producing germinal centres. The presence of TLS has been associated with improved patient outcome and response to immunotherapy, though the mechanisms remain unclear. Similarly, the contribution of B cells and TLS to anti-tumour immunity in non-small cell lung cancer (NSCLC) remains largely unexplored. Methods: We evaluated TRACERx and TCGA NSCLC cohorts to compare B cell and TLS signatures as well as BCR sequences from human adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC). Using a novel immunogenic mouse model of LUAD, we applied lineage tracing, 3D IF, and single-cell sequencing to characterise the evolution of lung germinal centre and antibody responses over tumour progression and immunotherapy treatment. We validated immunotherapy observations in a cohort of melanoma patients with pre- and post-treatment samples. Results: We found that B cell and TLS gene set scores were significantly higher in LUAD compared to LUSC. We examined B cell signals in specific mutational backgrounds, and observed an increase in BCR CDR3 diversity and IgG class switch in the context of clonal TP53 mutation. We therefore investigated the mechanistic basis of B cells in LUAD using an immunogenic mouse model on a KRAS/TP53 background. We found that TLS organisation was triggered by CXCL13 and that exogenous CXCL13 treatment increased B cell infiltration and overall survival. Using serum transfer experiments, we demonstrated that tumour-binding class-switched antibodies played an anti-tumour role and increased survival. We found that immunotherapy increased germinal centre B cell responses and antibody quantity and quality. Using single-cell BCR sequencing of immunotherapy-treated mice, we showed clonal expansion of B cells reactive to re-expressed endogenous retroviral antigens. Furthermore, we found that KRAS G12C but not MEK inhibition boosted B cell and antibody responses. Finally, combined treatment with CXCL13 and checkpoint blockade improved survival, suggesting that boosting local B cell responses can improve immunotherapy outcome. In checkpoint blockade-treated melanoma patients, we found that BCR diversity and the number of unique antigen specificities was elevated in responders post-therapy. Non-responders showed a loss of expanded clones, suggesting that both maintenance of expanded clones and recruitment of new clones associate with successful response. Conclusions: Our work describes a novel role for B cells in the lung tumour microenvironment and establish that TLS directly contribute to anti-tumour immunity by producing class-switched protective antibodies. We demonstrate that selective boosting of anti-tumour B cells shows therapeutic potential in combination with targeted and immunotherapy. Citation Format: Katey Enfield, Kevin Ng, Jesse Boumelha, Kevin Litchfield, Oriol Pich, TRACERx Consortium, Charles Swanton, George Kassiotis, Julian Downward. Immunotherapy boosts B cell responses in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1390.