Induce Tumor Regression Research Articles

Celecoxib induces adipogenic differentiation of hemangioma-derived mesenchymal stem cells through the PPAR-γ pathway in vitro and in vivo.

Infantile hemangioma (IH) is a benign tumor that produces a permanent scar or a mass of fibro-fatty tissue after involution in 40-80% of cases. Celecoxib is an inhibitor of cyclooxygenase-2 (COX-2), and can inhibit angiogenesis and fibrosis. The present study aimed to clarify whether celecoxib is able to induce tumor regression with minimal side effects. For that purpose, the regulation of celecoxib in the involution of IH was investigated in an IH model. Hemangioma-derived mesenchymal stem cells (Hem-MSCs) were isolated from proliferating specimens, and an IH model was established by injecting these cells into nude mice. Celecoxib was administered in vitro and in vivo. Oil Red O staining and reverse transcription-quantitative-PCR were used to detect the adipogenic differentiation of Hem-MSCs. Histologic analysis and immunohistochemical staining of the tumor xenografts were performed to investigate the pathological evolution of the tumor. The results showed that celecoxib inhibited the proliferation and induced the adipogenic differentiation of Hem-MSCs in vitro. In vivo, adipocytes were only present in the celecoxib group at week 4, while a larger number of fibroblasts and collagenous fibers could be observed in the basic fibroblast growth factor group. Therefore, celecoxib may be a potential agent used for IH treatment by inducing adipogenesis and inhibiting fibroblast formation.

Developing a translational murine-to-canine pathway for an IL-2/agonist anti-CD40 antibody cancer immunotherapy.

Human and canine sarcomas are difficult to treat soft tissue malignancies with an urgent need for new improved therapeutic options. Local recurrence rates for humans are between 10%–30%, and 30%–40% develop metastases. Outcomes for dogs with sarcoma vary with grade but can be similar. Pet dogs share the human environment and represent human cancer with genetic variation in hosts and tumours. We asked if our murine studies using genetically identical mice and cloned tumour cells were translatable to larger, genetically diverse domestic dogs with naturally occurring tumours, to (i) develop a canine cancer therapeutic, and (ii) to use as a translational pathway to humans. Our murine studies showed that intra‐tumoral delivery of interleukin‐2 (IL‐2) plus an agonist anti‐CD40 antibody (Ab) induces long‐term curative responses ranging from 30% to 100%, depending on tumour type. We developed an agonist anti‐canine‐CD40 Ab and conducted a phase I dose finding/toxicology 3 + 3 clinical trial in dogs (n = 27) with soft tissue sarcomas on account of suitability for intratumoral injection and straightforward monitoring. Dogs were treated with IL‐2 plus anti‐CD40 antibody for 2 weeks. Three dose levels induced tumour regression with minimal side effects, measured by monitoring, haematological and biochemical assays. Importantly, our mouse and canine studies provide encouraging fundamental proof‐of‐concept data upon which we can develop veterinary and human immunotherapeutic strategies.

PKHB1, a thrombospondin-1 peptide mimic, induces anti-tumor effect through immunogenic cell death induction in breast cancer cells

ABSTRACT Breast cancer is the most commonly diagnosed cancer and the leading cause of cancer death in women worldwide. Recent advances in the field of immuno-oncology demonstrate the beneficial immunostimulatory effects of the induction of immunogenic cell death (ICD). ICD increases tumor infiltration by T cells and is associated with improved prognosis in patients affected by triple negative breast cancer (TNBC) with residual disease. The aim of this study was to evaluate the antitumoral effect of PKHB1, a thrombospondin-1 peptide mimic, against breast cancer cells, and the immunogenicity of the cell death induced by PKHB1 in vitro, ex vivo, and in vivo. Our results showed that PKHB1 induces mitochondrial alterations, ROS production, intracellular Ca2+ accumulation, as well calcium-dependent cell death in breast cancer cells, including triple negative subtypes. PKHB1 has antitumor effect in vivo leading to a reduction of tumor volume and weight and promotes intratumoral CD8 + T cell infiltration. Furthermore, in vitro, PKHB1 induces calreticulin (CALR), HSP70, and HSP90 exposure and release of ATP and HMGB1. Additionally, the killed cells obtained after treatment with PKHB1 (PKHB1-KC) induced dendritic cell maturation, and T cell antitumor responses, ex vivo. Moreover, PKHB1-KC in vivo were able to induce an antitumor response against breast cancer cells in a prophylactic application, whereas in a therapeutic setting, PKHB1-KC induced tumor regression; both applications induced a long-term antitumor response. Altogether our data shows that PKHB1, a thrombospondin-1 peptide mimic, has in vivo antitumor effect and induce immune system activation through immunogenic cell death induction in breast cancer cells.

A non-coding RNA balancing act: miR-346-induced DNA damage is limited by the long non-coding RNA NORAD in prostate cancer

BackgroundmiR-346 was identified as an activator of Androgen Receptor (AR) signalling that associates with DNA damage response (DDR)-linked transcripts in prostate cancer (PC). We sought to delineate the impact of miR-346 on DNA damage, and its potential as a therapeutic agent.MethodsRNA-IP, RNA-seq, RNA-ISH, DNA fibre assays, in vivo xenograft studies and bioinformatics approaches were used alongside a novel method for amplification-free, single nucleotide-resolution genome-wide mapping of DNA breaks (INDUCE-seq).ResultsmiR-346 induces rapid and extensive DNA damage in PC cells - the first report of microRNA-induced DNA damage. Mechanistically, this is achieved through transcriptional hyperactivation, R-loop formation and replication stress, leading to checkpoint activation and cell cycle arrest. miR-346 also interacts with genome-protective lncRNA NORAD to disrupt its interaction with PUM2, leading to PUM2 stabilisation and its increased turnover of DNA damage response (DDR) transcripts. Confirming clinical relevance, NORAD expression and activity strongly correlate with poor PC clinical outcomes and increased DDR in biopsy RNA-seq studies. In contrast, miR-346 is associated with improved PC survival.INDUCE-seq reveals that miR-346-induced DSBs occur preferentially at binding sites of the most highly-transcriptionally active transcription factors in PC cells, including c-Myc, FOXA1, HOXB13, NKX3.1, and importantly, AR, resulting in target transcript downregulation. Further, RNA-seq reveals widespread miR-346 and shNORAD dysregulation of DNA damage, replication and cell cycle processes.NORAD drives target-directed miR decay (TDMD) of miR-346 as a novel genome protection mechanism: NORAD silencing increases mature miR-346 levels by several thousand-fold, and WT but not TDMD-mutant NORAD rescues miR-346-induced DNA damage. Importantly, miR-346 sensitises PC cells to DNA-damaging drugs including PARP inhibitor and chemotherapy, and induces tumour regression as a monotherapy in vivo, indicating that targeting miR-346:NORAD balance is a valid therapeutic strategy.ConclusionsA balancing act between miR-346 and NORAD regulates DNA damage and repair in PC. miR-346 may be particularly effective as a therapeutic in the context of decreased NORAD observed in advanced PC, and in transcriptionally-hyperactive cancer cells.

Discovery of a Conditionally Activated IL-2 that Promotes Antitumor Immunity and Induces Tumor Regression.

IL-2 is a cytokine clinically approved for the treatment of melanoma and renal cell carcinoma. Unfortunately, its clinical utility is hindered by serious side effects driven by the systemic activity of the cytokine. Here, we describe the design and characterization of a conditionally activated IL-2 prodrug, WTX-124, that takes advantage of the dysregulated protease milieu of tumors. WTX-124 was engineered as a single molecule containing an inactivation domain and a half-life extension domain that are tethered to a fully active IL-2 by protease-cleavable linkers. We show that the inactivation domain prevented IL-2 from binding to its receptors in nontumor tissues, thereby minimizing the toxicity associated with systemic exposure to IL-2. The half-life extension element improves the pharmacokinetic profile of WTX-124 over free IL-2, allowing for greater exposure. WTX-124 was preferentially activated in tumor tissue by tumor-associated proteases, releasing active IL-2 in the tumor microenvironment. In vitro assays confirmed that the activity of WTX-124 was dependent on proteolytic activation, and in vivo WTX-124 treatment resulted in complete rejection of established tumors in a cleavage-dependent manner. Mechanistically, WTX-124 treatment triggered the activation of T cells and natural killer (NK) cells, and markedly shifted the immune activation profile of the tumor microenvironment, resulting in significant inhibition of tumor growth in syngeneic tumor models. Collectively, these data demonstrate that WTX-124 minimizes the toxicity of IL-2 treatment in the periphery while retaining the full pharmacology of IL-2 in the tumor microenvironment, supporting its further development as a cancer immunotherapy treatment. See related Spotlight by Silva, p. 544.

Combined inhibition of HMGCoA reductase and mitochondrial complex I induces tumor regression of BRAF inhibitor-resistant melanomas

BackgroundPrimary and posttreatment resistance to BRAFV600 mutation–targeting inhibitors leads to disease relapse in a majority of melanoma patients. In many instances, this resistance is promoted by upregulation of mitochondrial oxidative phosphorylation (OxPhos) in melanoma cells. We recently showed that a novel electron transport chain (ETC) complex I inhibitor, IACS-010759 (IACS), abolished OxPhos and significantly inhibited tumor growth of high-OxPhos, BRAF inhibitor (BRAFi)–resistant human melanomas. However, the inhibition was not uniform across different high OxPhos melanomas, and combination with BRAFi did not improve efficacy.MethodsWe performed a high-throughput unbiased combinatorial drug screen of clinically relevant small molecules to identify the most potent combination agent with IACS for inhibiting the growth of high-OxPhos, BRAFi-resistant melanomas. We performed bioenergetics and carbon-13 metabolite tracing to delineate the metabolic basis of sensitization of melanomas to the combination treatment. We performed xenograft tumor growth studies and Reverse-Phase Protein Array (RPPA)–based functional proteomics analysis of tumors from mice fed with regular or high-fat diet to evaluate in vivo molecular basis of sensitization to the combination treatment.ResultsA combinatorial drug screen and subsequent validation studies identified Atorvastatin (STN), a hydroxymethylglutaryl-coenzyme A reductase inhibitor (HMGCRi), as the most potent treatment combination with IACS to inhibit in vitro cell growth and induce tumor regression or stasis of some BRAFi-resistant melanomas. Bioenergetics analysis revealed a dependence on fatty acid metabolism in melanomas that responded to the combination treatment. RPPA analysis and carbon-13 tracing analysis in these melanoma cells showed that IACS treatment decreased metabolic fuel utilization for fatty acid metabolism, but increased substrate availability for activation of the mevalonate pathway by HMGCR, creating a dependence on this pathway. Functional proteomic analysis showed that IACS treatment inhibited MAPK but activated AKT pathway. Combination treatment with STN counteracted AKT activation.ConclusionsSTN and other clinically approved HMGCRi could be promising combinatorial agents for improving the efficacy of ETC inhibitors like IACS in BRAFi-resistant melanomas.

An Orally Bioavailable and Highly Efficacious Inhibitor of CDK9/FLT3 for the Treatment of Acute Myeloid Leukemia.

Simple SummaryCDDD11-8 is an orally bioactive pharmacological inhibitor of CDK9. In AML cells, CDDD11-8 suppressed the proliferation and caused robust inhibition of tumor growth in vivo via synergistic inhibition of FLT3. The drug candidate has potential to overcome cancer cell resistance to FLT3 inhibition by concurrently blocking the CDK9-mediated upregulation of cancer cell-survival genes.Mutations in FMS-like tyrosine kinase 3 (FLT3) occur in approximately one-third of AML patients and are associated with a particularly poor prognosis. The most common mutation, FLT3-ITD, is a self-activating internal tandem duplication (ITD) in the FLT3 juxtamembrane domain. Many FLT3 inhibitors have shown encouraging results in clinical trials, but the rapid emergence of resistance has severely limited sustainable efficacy. Co-targeting of CDK9 and FLT3 is a promising two-pronged strategy to overcome resistance as the former plays a role in the transcription of cancer cell-survival genes. Most prominently, MCL-1 is known to be associated with AML tumorigenesis and drug resistance and can be down-regulated by CDK9 inhibition. We have developed CDDD11-8 as a potent CDK9 inhibitor co-targeting FLT3-ITD with Ki values of 8 and 13 nM, respectively. The kinome selectivity has been confirmed when the compound was tested in a panel of 369 human kinases. CDDD11-8 displayed antiproliferative activity against leukemia cell lines, and particularly potent effects were observed against MV4-11 and MOLM-13 cells, which are known to harbor the FLT3-ITD mutation and mixed lineage leukemia (MLL) fusion proteins. The mode of action was consistent with inhibition of CDK9 and FLT3-ITD. Most importantly, CDDD11-8 caused a robust tumor growth inhibition by oral administration in animal xenografts. At 125 mg/kg, CDDD11-8 induced tumor regression, and this was translated to an improved survival of animals. The study demonstrates the potential of CDDD11-8 towards the future development of a novel AML treatment.

Abstract P5-16-18: Developing IpY: A novel inhibitor for the treatment of ER+ CDK4i-resistant breast cancer

Abstract Almost 270,000 US women will be diagnosed with breast cancer (BC) this year, anddespite advances in treatment, ~40,000 women will die. ER/PR+ (endocrineresponsive), Her2- tumors occur in approximately 40% of breast cancer patients andare candidates for drugs targeting estrogen responsiveness, such as letrozole orfulvestrant. CDK4 targeting drugs (CDK4i) like palbociclib, abemaciclib, or ribociclib arenow approved in combination with letrozole or fulvestrant as a front- and second-linetherapy for metastatic ER/PR+, Her2- patients. However, while combined ER andCDK4i treatment significantly extends Progression Free Survival (PFS), those treatedinvariably develop resistance. Thus, drug resistance remains an urgent unmet need, as current treatments only marginally improve Overall Survival (OS) for these patients.Resistance to palbociclib develops because of compensation by another kinase, CDK2, suggesting that to be effective, therapies must be developed to inhibit both CDK4 andCDK2. Concarlo has taken a different approach to inhibit CDK4 and CDK2 by targetingp27Kip1 (p27). p27 interacts specifically with CDK4/6 and CDK2, and is responsible forturning these kinases ON and OFF. This transition from ON to OFF is mediated by aspecific modification to p27 itself, by the tyrosine kinase BRK (breast tumor relatedkinase). Published work has shown that a naturally occurring ALTernatively splicedform of BRK, ALT, can bind to p27, blocking BRK's association, and preventing BRK'sphosphorylation of p27. This locks CDK4 and CDK2 into the OFF conformation andsimultaneously inhibits activities of both kinases. As the naked 144 aa ALT peptidedoes not enter cells, it was formulated within a lipid nano-particle (NP), and called IpY.1.IpY.1 inhibits proliferation of HR+ and triple negative (TN) BC cells, but not non-cancerous breast cells MCF10A. IpY.1 arrests CDK4i-resistant and endocrine-resistantBC cells and it is both cytostatic and cytotoxic, demonstrating that p27 is a viabletherapeutic target to combat drug resistance. In vivo, IpY reduces tumor volumes and increase OS in cell line-derived xenografts (CDX). In order to convert manufacturing of the therapeutic peptide portion of IpY from recombinant to synthetic production, Concarlo truncated the large ALT peptide and bioengineered it to a more stable form. IpY.20 has a comparable IC50 as IpY.1 in HR+and TNBC cells and does not cause growth arrest in non-cancerous MCF10A cells. Using the fluorescent-labeled variant of IpY.20 (fluo-IpY.20) injected into tumor-bearingsyngeneic CDX model, followed by IVIS imaging, we could detect fluo-IpY.20 in tumorsas early as 15 min and up to 24h post injection. 6-10% of the total fluorescent signal isallocated to tumor at all timepoints. In addition, fluo-IpY.20 inhibits the activation of itstarget in tumors within 24h, suggesting fluo-IpY.20 not only reaches tumors but alsoengages with its target. IpY.20 induces tumor regression in CDX models and animmunocompetent genetically engineered model that overexpresses the potent Erbb2oncogene. Overall, repeated dosing of IpY.20 does not exhibit significant adverseeffects and is well tolerated in immunocompetent mice. However, in these treated mice, there was an increase in platelets and production of some cytokines compared to vehicle-treated mice, suggesting that an immune response may have been initiated. By targeting p27 instead of the conserved CDK4 or CDK2 active sites, IpY.20 will bemore selective and have fewer off-target effects than the ATP-competitive smallmolecule CDK4is currently in use. As the majority of the drug resistance seen withCDK4 inhibitory drugs is a result of compensatory CDK2 activity, IpY.20 preventsacquired drug resistance by hitting both CDK4 and CDK2 simultaneously, resulting in amore durable response to the drug. Concarlo is currently in manufacturing with IpY.20. Citation Format: Grace Chen, Stacy W Blain, Irina Jilishitz, Allison Vanlnwegen, Lingyue Yan, Yun Wu. Developing IpY: A novel inhibitor for the treatment of ER+ CDK4i-resistant breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-16-18.

Abstract OT2-23-01: A Phase 1b/2a, open-label platform study to evaluate mirdametinib in combination with fulvestrant in ER+ metastatic breast cancers harboring MAPK-activating mutations

Abstract Background: Approximately 70% of all metastatic breast cancers (MBCs) express estrogen receptor (ER). Hormonal therapies targeting ER are highly active against these cancers and have been remarkably successful in improving outcomes. Unfortunately, resistance to hormonal therapy is nearly universal and over 90% of patients develop resistance to various drugs targeting ER. We have identified mutations in three non-overlapping gene sets that are associated with the hormone-resistant phenotype: (1) ESR1, (2) MAPK pathway, and (3) transcription factors. The finding of mutations that might activate MAPK signaling was particularly striking given the known oncogenic function of this pathway in other cancers and the potential to target this pathway with selective inhibitors. Indeed, in preclinical models, the combination of an allosteric MEK inhibitor with an ER antagonist induced tumor regression in NF1-null, ER+ xenografts that were resistant to antiestrogen monotherapy. These data suggest that MAPK pathway alterations promote resistance to ER-targeted therapies in MBC and lead us to hypothesize that MAPK-targeted therapies will prove highly effective in such patients. However, NF1 (prevalence 6%) is only one of several RAS modulators that are associated with hormone resistance, and KRAS and HRAS mutations were also present in this dataset. Further, receptor tyrosine kinase (RTK) alterations (EGFR amplification and ERBB2 somatic mutations) and mutations in the RAF/MEK kinase cascade (BRAF and MEK1) are present and may comparably confer resistance to hormonal therapy and sensitivity to MAPK pathway inhibition. We hypothesize that MAPK pathway-activated MBCs can be effectively treated using MAPK targeted therapies in combination with hormonal therapy. Patient Accrual:The workflow to enroll patients to clinical trials at MSKCC is automated such that genomic alterations that are deposited on the cBioPortal are matched with appropriate clinical trials using database queries. A recent query of cBioPortal data from ER+ MBC patients treated at MSKCC showed 216 patients harboring actionable alterations in the MAPK pathway. Clinical Trial Design: This investigator-initiated trial being performed in collaboration with SpringWorks Therapeutics is IRB-approved and anticipated to open at MSKCC in August 2021. This is a Phase IB/IIA clinical trial of the allosteric MEK1/2 inhibitor mirdametinib (oral, daily) with the ER antagonist fulvestrant (500mg IM monthly). Inclusion criteria for the study will include ER+ tumor (ASCO-CAP guidelines), MAPK-activating genomic alteration documented by a CLIA-certified NGS assay at any time before the start of treatment, measurable disease, and at least one line of prior endocrine therapy. The feasibility of the combination will be established in a brief safety run-in of full-dose fulvestrant together with mirdametinib with dose de-escalation planned if two Dose Limiting Toxicities (DLT) are observed in the first six patients. This drug combination will then be expanded in three cohorts of patients defined by tumor genotype: (1) RAS activating: NF1 loss, KRAS or HRAS activating mutation, (2) RTK: EGFR amplification or ERBB2 hotspot mutation, (3) RAF/MEK: activating mutation in BRAF, CRAF, or MEK1/2. The primary endpoint for the study will be safety and tolerability of mirdametinib in combination with fulvestrant, and secondary endpoints include Overall Response Rate (ORR), Progression Free Survival (PFS), Clinical Benefit Rate (CBR), and Duration of Response (DOR) for the drug combination in each cohort and for the total study population. Overall survival is not an endpoint of this protocol. Citation Format: Ezra Y. Rosen, Payal Patel, Nenad Sarapa, Badreddin Edris, Alexia Iasonos, Komal Jhaveri, Pedram Razavi, Mark Robson, Alexander Drilon, Michael F. Berger, David B. Solit, Sarat Chandarlapaty. A Phase 1b/2a, open-label platform study to evaluate mirdametinib in combination with fulvestrant in ER+ metastatic breast cancers harboring MAPK-activating mutations [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr OT2-23-01.

CAR NK-92 cells targeting DLL3 kill effectively small cell lung cancer cells in vitro and in vivo.

Small cell lung cancer (SCLC) is characterized by a high relapse rate, drug tolerance, and limited treatment choices. Chimeric antigen receptor (CAR)-modified NK cells represent a promising immunotherapeutic modality for cancer treatment. However, their potential applications have not been explored in SCLC. Delta-like ligand 3 (DLL3) has been reported to be overexpressed in SCLC and may be a rational target for CAR NK immunotherapy. In this study, we developed DLL3-specific NK-92 cells and explored their potential in the treatment of SCLC. A coculture of DLL3+ SCLC cell lines with DLL3-CAR NK-92 cells exhibited significant in vitro cytotoxicity and cytokine production. DLL3-CAR NK-92 cells induced tumor regression in an H446-derived pulmonary metastasis tumor model under a good safety threshold. The potent antitumor activities of DLL3-CAR NK-92 cells were observed in subcutaneous tumor models of SCLC. Moreover, obvious tumor-infiltrated DLL3-CAR NK-92 cells were detected in DLL3+ SCLC xenografts. These findings indicate that DLL3-CAR NK-92 cells might be a potential strategy for the treatment of SCLC.

The combination therapy of Everolimus and anti-PD-1 improves the antitumor effect by regulating CD8+ T cells in bladder cancer.

This study aimed to investigate the efficacy of Everolimus (EVE) in combination with immune checkpoint inhibitors (ICIs) in bladder cancer treatment and the underlying mechanisms. In vitro, MB49 cells were exposed to gradient concentrations (0nM-100nM) of EVE for 48h, to investigate the cell viability and cell proliferative potential. In vivo, we applied a subcutaneous tumor mouse model of bladder cancer and the mice were treated with EVE monotherapy (different doses) or in combination with anti-programmed cell death protein 1 (PD-1) agents to study the impacts on tumor growth and explore the immune mechanism. The influences of treatments on peripheral immune profiles and tumor immune microenvironment were also discussed. EVE could inhibit the growth of MB49 cells in vitro. Though high-dose EVE monotherapy could induce tumor regression in vivo, it also contributed to immunosuppression. High-dose EVE inhibited the expression of PD-L1 by inhibiting Th1 cytokine secretion, while combined therapy with PD-1 inhibitors showed no extra profit. Low-dose EVE in combination with PD-1 inhibitors could effectively suppress tumor growth by increasing periphery CD8+ T cell frequency and GZMB+ CD8+ T cell frequency in the tumor microenvironment. High-dose EVE monotherapy induced tumor regression, but with immunosuppression to some content. Combination therapy with low-dose EVE and PD-1 inhibitor could effectively inhibit the growth of bladder tumors by enhancing the antitumor immunity of CD8+ T cells in both periphery and tumor microenvironment.

Glutamine Antagonist Synergizes with Electrodynamic Therapy to Induce Tumor Regression and Systemic Antitumor Immunity.

Electrodynamic therapy (EDT) combining nanotechnology with electronic current was used in this study to generate highly cytotoxic oxidative hydroxyl radicals (·OH) for tumor destruction. However, increasing evidence suggests that EDT treatment alone for one time still faces great challenges in achieving long-term tumor suppression in an immunosuppressive environment, which would raise the risk of later tumor recurrence. Benefitting from the marvelous potential of reactive oxygen species (ROS)-mediated dynamic therapies in tumor immunocombination therapy due to their immunogenic cell death (ICD) effect, a glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON)-loaded nanocarrier (Pt-Pd@DON) was designed for combination therapy (EDT and immunotherapy) against tumor recurrence and metastasis. The protective immune response was motivated in highly immunosuppressive tumors by the joint functions of ICD and CD8+ T cell infiltration promoted by DON. A great therapeutic efficacy has been demonstrated in primary and metastatic tumor models, respectively. This study has provided an effective thought way for clinical highly immunosuppressive tumor treatment.

Allogeneic Tumor Antigen-Specific T Cells for Broadly Applicable Adoptive Cell Therapy of Cancer.

T cells specific for major histocompatibility complex (MHC)-presented tumor antigens are capable of inducing durable remissions when adoptively transferred to patients with refractory cancers presenting such antigens. When such T cells are derived from healthy donors, they can be banked for off-the-shelf administration in appropriately tissue matched patients. Therefore, tumor antigen-specific, donor-derived T cells are expected to be a mainstay in the cancer immunotherapy armamentarium. In this chapter, we analyze clinical evidence that tumor antigen-specific donor-derived T cells can induce tumor regressions when administered to appropriately matched patients whose tumors are refractory to standard therapy. We also delineate the landscape of MHC-presented and unconventional tumor antigens recognized by T cells in healthy individuals that have been targeted for adoptive T cell therapy, as well as emerging antigens for which mounting evidence suggests their utility as targets for adoptive T cell therapy. We discuss the growing technological advancements that have facilitated sequence identification of such antigens and their cognate T cells, and applicability of such technologies in the pre-clinical and clinical settings.

Treatment with b-AP15 to Inhibit UCHL5 and USP14 Deubiquitinating Activity and Enhance p27 and Cyclin E1 for Tumors with p53 Deficiency.

Background: TP53 protein is lost or mutated in about half of all types of human cancers and small molecules to regulate mutant p53 repair, or interrupt ubiquitination degradation of p53 induced by E3-ubiquitin ligase Mdm2 have a potential application in clinical application. Methods: To inhibit the deubiquitinase activity of 19S proteasome and restore the p53 protein level, in this study, we utilized p53 knockout mice to test the anti-cancer effect of a specific USP14 and UCH37 inhibitor b-AP15. Results: Our results show that UCHL5, USP14 and COPS5 are upregulated in p53-related tumors, and higher expression of these genes results in a shorter overall survival in patients with p53 deficiency. Treatment with b-AP15, a UCHL5 and USP14 deubiquitinating activity inhibitor in 19S regulatory subunit, induces tumor regression and prolong the survival period of tumor-loaded mice through down-regulation of COPS5 and its downstream AP-1 and E2F1, and up-regulation of the cell cycle-related proteins p27 and Cyclin E1. Conclusions: Thus, our results suggested that inhibition of UCHL5 and USP14 deubiquitinating activity in 19S proteasome may contribute an extensive approach to preventing tumor progress due to p53 deficiency.

Neoadjuvant programmed cell death 1 blockade combined with chemotherapy for resectable esophageal squamous cell carcinoma

BackgroundProgrammed cell death 1 (PD-1) blockade induces tumor regression in patients with advanced esophageal squamous cell carcinoma (ESCC); however, little is known about the efficacy of PD-1 blockade as neoadjuvant...

Hypoxia induction of SH2D3A triggers malignant progression of lung cancer.

Lung cancer is the most prevalent and aggressive cancer and is one of the leading causes of cancer-related death worldwide. Hypoxia in the tumor microenvironment is associated with poor patient survival and is a crucial characteristic of solid tumors. A subset of tumor cells, termed cancer stem cells (CSCs), with self-renewal and differentiation capabilities simultaneously, are regarded as responsible for cancer tumorigenesis, resistance to therapeutics, and cancer relapse. Recent advances have revealed that hypoxia plays an essential role in CSCs self-replication maintenance. Yet, the underlying mechanisms of hypoxia that trigger the stemness maintenance of CSCs are still poorly understood. Here, we provide evidence showing that SH2D3A expression level was increased in lung cancer and lung CSCs, and high expression of SH2D3A was associated with the overall survival of patients with lung cancer. Mechanistically, HIF-2α, which is a key transcription factor in response to hypoxia directly binds to the SH2D3A promoter and facilitates SH2D3A expression at the transcription level. SH2D3A was found to be functionally important for lung CSC malignant behaviors such as uncontrolled self-replication and proliferation. We demonstrated that pharmacological downregulation of SH2D3A expression by AM966, a small molecule compound, efficiently induces tumor regression in vitro and in vivo. Thus, this study highlights the biological implications of SH2D3A as a novel prognostic marker and therapeutic target in lung cancer in the future.

Determining PD-L1 expression in head and neck squamous cell carcinoma using immunohistochemistry.

Advanced head and neck squamous cell carcinoma (HNSCC) has limited treatment options. Programmed death-ligand1 (PD-L1) expressed by tumor cells interacts with PD-1 receptor on T lymphocytes leading to immune evasive response and survival advantage. Therapy with immune check-point inhibitors target PD-1/PD-L1 blockade inducing tumor regression. Immunohistochemistry (IHC) for PD-L1 expression enables patient selection for immunotherapy and may be considered a potential predictor of clinical response. A retrospective analysis of IHC for PD-L1 expression using manual laboratory developed technique (LDT) with antibody clone 22C3 (Dako) in 93 cases of HNSCC. PD-L1 expression was correlated with age, gender, tumor site, grade and stage. PD-L1 IHC was performed in 93 cases and immunopositivity was noted in 59 (63.4%) cases. High expression with combined proportion score (CPS) ≥50 was seen in 15 (16.1%) cases and low expression with CPS ≥1 expression was seen in 44 (47.3%) cases. An almost-perfect interobserver agreement was noted by two pathologists for PD-L1 IHC expression (Cohen's kappa coefficient = 0.910). No statistically significant correlation was noted between PD-L1 score and patient demographics, tumor site, grade or stage. Detection of PD-L1 status by IHC enables identification of HNSCC patients eligible for future targeted immunotherapy.

IL-2 in combination with immune checkpoints inhibitors as a promising approach for cancer immunotherapy: A literature review

Relevance: Interleukin-2 (IL-2) alone has been shown to induce tumor regression and approved to treat metastatic renal cancer and melanoma. Checkpoint inhibitors realize their therapeutic effect through stimulation of immune system effectors. One of such mechanisms is the enhancement of IL-2 production by T-helpers.
 The purpose of the study was to determine the effectiveness of IL-2 administration as a component of combined immunotherapy with immune checkpoint inhibitors and to suggest the mechanisms by which IL-2 can reduce the frequency and severity of side effects during checkpoint inhibitor therapy without reducing their effectiveness.
 Methods: The literature search was performed in the PubMed, SCOPUS, and Web of Science databases by the keywords in article titles: “immunotherapy,” “checkpoint inhibitors,” “interleukin-2,” and “combination therapy” for the period 2011-2021.
 A total of 248 relevant articles were found. The review’s inclusion criteria were: clinical cases; data of clinical research methods; data on humans/body fluids from humans; literature reviews and meta-analyses. The selected 24 articles met the search criteria and were included in the study.
 Results: The combined action of IL-2 and сheckpoint inhibitors increases the proliferative and killer activity of the antitumor immunity effectors compared to the action of the same drugs in mono-mode at a level exceeding the summation effect.
 Conclusion: The combination of IL-2 and сheckpoint inhibitors can increase the effectiveness of anticancer treatment and is a promising area of immunotherapy

Il-2 in combination with immune checkpoints
 inhibitors as a promising approach for cancer
 immunotherapy:
 A literature review

Relevance: Interleukin-2 (IL-2) alone has been shown to induce tumor regression and approved to treat metastatic renal cancer and melanoma. Checkpoint inhibitors realize their therapeutic
 effect through stimulation of immune system effectors; one of such
 mechanisms is the enhancement of IL-2 production by T-helpers.
 The purpose of the study was to determine the effectiveness
 of IL-2 administration as a component of combined immunotherapy with immune checkpoint inhibitors and to suggest the mechanisms by which IL-2 can reduce the frequency and severity of side
 effects during checkpoint inhibitor therapy without reducing their
 effectiveness.
 Methods: The literature search was performed in the PubMed,
 SCOPUS, and Web of Science databases by the keywords in article titles: “immunotherapy,” “checkpoint inhibitors,” “interleukin-2,” and
 “combination therapy” for the period 2011-2021. A total of 248 relevant articles were found. The review’s inclusion criteria were: clinical
 cases; data of clinical research methods; data on humans/body fluids from humans; literature reviews and meta-analyses. The selected
 24 articles met the search criteria and were included in the review.
 Results: The combined action of IL-2 and сheckpoint inhibitors increases the proliferative and killer activity of the antitumor
 immunity effectors compared to the action of the same drugs in
 mono-mode at a level exceeding the summation effect.
 Conclusion: The combination of IL-2 and сheckpoint inhibitors can increase the effectiveness of anticancer treatment and is
 a promising area of immunotherapy

Abstract P237: PRT2527 is a potent and selective CDK9 inhibitor that demonstrates anti-cancer activity in preclinical models of hematological malignancies and solid tumors with MYC amplification

Abstract Cyclin-dependent kinase 9 (CDK9) is a master regulator of transcription that controls paused RNA polymerase II (RNAP2) release through phosphorylation of its carboxy-terminal domain, resulting in productive transcription elongation. CDK9 has been extensively studied as a potential target for cancer therapy in “transcriptionally addicted” tumors as transient inhibition of CDK9 primarily depletes proteins with short half-lives, such as the oncogenes MCL1 and MYC, making CDK9 a promising target in cancer. Here we show that PRT2527 is a potent and highly selective CDK9 inhibitor with moderate to high clearance that achieves optimal temporal target engagement and exhibits potent in vitro and in vivo activities. PRT2527 inhibited CDK9 enzymatic activity with an IC50 of 0.98 nM in a biochemical assay and showed high selectivity in a panel of kinases when tested at physiologically relevant 1 mM ATP concentration. In vitro, PRT2527 inhibited phosphorylation of Ser2RNAP2 in NCI-H929 cells with an IC50 of 54 nM, and an IC50 of 198 nM in a plasma assay to adjust for human plasma protein binding. Transient treatment of cells with PRT2527 inhibited pSer2RNAP2, depleted MCL1 and MYC proteins, and activated cleaved caspase-3 (CC3) in a concentration-dependent manner. In a proteomic profiling study, MCL1 was identified as one of the major down-regulated proteins following PRT2527 treatment. In a panel of hematological cancer cell lines representing B- and T-ALL, AML, and non-Hodgkin’s lymphoma (NHL), as well as subsets of sarcoma, prostate, adenoid cystic carcinoma (ACC), and non-small cell lung cancer (NSCLC) cell lines, PRT2527 treatment consistently led to a potent, concentration-dependent inhibition of proliferation. In a pharmacokinetic/pharmacodynamic (PK/PD) study, intravenous (IV) administration of PRT2527 achieved transient target engagement, depletion of MCL1 and MYC proteins, and induction of apoptosis in tumor tissue. This PK/PD correlation was successfully translated into in vivo efficacy in multiple models. Once weekly dosing of PRT2527 was well-tolerated and significantly inhibited tumor growth in various AML CDX models and induced tumor regressions in double-hit and triple-hit diffuse large B-cell lymphoma (DLBCL) CDX and PDX models carrying the MYC translocation. Combining PRT2527 with venetoclax achieved complete tumor regressions in a venetoclax resistant OCI-AML3 model. PRT2527 demonstrated potent ex vivo activity in PDX models of B-ALL and T-ALL, as well as various solid tumor PDX models with high levels of MYC amplification and overexpression, including pancreatic carcinoma, gastric and gastroesophageal carcinomas, NSCLC, and sarcoma. In vivo efficacy studies with once weekly IV administration of PRT2527 confirmed significant tumor growth inhibition in select MYC-amplified solid tumor PDX models. Taken together, this preclinical characterization supports the advancement of PRT2527 into clinical studies for transcriptionally addicted hematological malignancies and solid tumors with MYC amplification and/or dysregulation. Citation Format: Yang W. Zhang, Liang Lu, Min Wang, Dave Rominger, Stefan Ruepp, Kirsten Gallagher, William Gowen-MacDonald, Chaofeng Dai, Miles Cowart, Andrew Combs, Bruce Ruggeri, Peggy Scherle, Kris Vaddi. PRT2527 is a potent and selective CDK9 inhibitor that demonstrates anti-cancer activity in preclinical models of hematological malignancies and solid tumors with MYC amplification [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P237.