Increased Tumor Growth Research Articles

Quantitative modeling of tumor dynamics and development of drug resistance in non-small cell lung cancer patients treated with erlotinib.

Insight into the development of treatment resistance can support the optimization of anticancer treatments. This study aims to characterize the tumor dynamics and development of drug resistance in patients with non-small cell lung cancer treated with erlotinib, and investigate the relationship between baseline circulating tumor DNA (ctDNA) data and tumor dynamics. Data obtained for the analysis included (1) intensively sampled erlotinib concentrations from 29 patients from two previous pharmacokinetic (PK) studies, and (2) tumor sizes, ctDNA measurements, and sparsely sampled erlotinib concentrations from 18 patients from the START-TKI study. A two-compartment population PK model was first developed which well-described the PK data. The PK model was subsequently applied to investigate the exposure-tumor dynamics relationship. To characterize the tumor dynamics, models accounting for intra-tumor heterogeneity and acquired resistance with or without primary resistance were investigated. Eventually, the model assumed acquired resistance only resulted in an adequate fit. Additionally, models with or without exposure-dependent treatment effect were explored, and no significant exposure-response relationship for erlotinib was identified within the observed exposure range. Subsequently, the correlation of baseline ctDNA data on EGFR and TP53 variants with tumor dynamics' parameters was explored. The analysis indicated that higher baseline plasma EGFR mutation levels correlated with increased tumor growth rates, and the inclusion of ctDNA measurements improved model fit. This result suggests that quantitative ctDNA measurements at baseline have the potential to be a predictor of anticancer treatment response. The developed model can potentially be applied to design optimal treatment regimens that better overcome resistance.

The IL6/JAK/STAT3 signaling axis is a therapeutic vulnerability in SMARCB1-deficient bladder cancer

SMARCB1 loss has long been observed in many solid tumors. However, there is a need to elucidate targetable pathways driving growth and metastasis in SMARCB1-deficient tumors. Here, we demonstrate that SMARCB1 deficiency, defined as genomic SMARCB1 copy number loss associated with reduced mRNA, drives disease progression in patients with bladder cancer by engaging STAT3. SMARCB1 loss increases the chromatin accessibility of the STAT3 locus in vitro. Orthotopically implanted SMARCB1 knockout (KO) cell lines exhibit increased tumor growth and metastasis. SMARCB1-deficient tumors show an increased IL6/JAK/STAT3 signaling axis in in vivo models and patients. Furthermore, a pSTAT3 selective inhibitor, TTI-101, reduces tumor growth in SMARCB1 KO orthotopic cell line-derived xenografts and a SMARCB1-deficient patient derived xenograft model. We have identified a gene signature generated from SMARCB1 KO tumors that predicts SMARCB1 deficiency in patients. Overall, these findings support the clinical evaluation of STAT3 inhibitors for the treatment of SMARCB1-deficient bladder cancer.

Heat shock protein gp96 drives natural killer cell maturation and anti-tumor immunity by counteracting Trim28 to stabilize Eomes

The maturation process of natural killer (NK) cells, which is regulated by multiple transcription factors, determines their functionality, but few checkpoints specifically targeting this process have been thoroughly studied. Here we show that NK-specific deficiency of glucose-regulated protein 94 (gp96) leads to decreased maturation of NK cells in mice. These gp96-deficient NK cells exhibit undermined activation, cytotoxicity and IFN-γ production upon stimulation, as well as weakened responses to IL-15 for NK cell maturation, in vitro. In vivo, NK-specific gp96-deficient mice show increased tumor growth. Mechanistically, we identify Eomes as the downstream transcription factor, with gp96 binding to Trim28 to prevent Trim28-mediated ubiquitination and degradation of Eomes. Our study thus suggests the gp96-Trim28-Eomes axis to be an important regulator for NK cell maturation and cancer surveillance in mice.

Cold-Inducible RNA Binding Protein Impedes Breast Tumor Growth in the PyMT Murine Model for Breast Cancer.

RNA binding proteins (RBPs) post-transcriptionally regulate gene expression by associating with regulatory sequences in the untranslated regions of mRNAs. Cold-inducible RBP (CIRP) is a stress-induced RBP that was recently shown to modulate inflammation in response to cellular stress, where it increases or decreases pro-tumorigenic (proinflammatory) cytokines in different contexts. CIRP expression is altered in several cancers, including breast cancer, but the effects of CIRP on inflammation in breast cancer is not known. Here, we investigate if CIRP alters growth and the inflammatory profile of breast tumors. Transgenic mice overexpressing CIRP in the mammary epithelium were crossed with the PyMT mouse model of breast cancer, and the effects on both early and late tumorigenesis and inflammation were assessed. The effects of CIRP knockdown were also assessed in Py2T cell grafts. Overexpression of CIRP led to decreased tumorigenesis in the PyMT mouse model. Conversely, the knockdown of CIRP in Py2T cell grafts led to increased tumor growth. Luminex cytokine assays assessed the effects on the inflammatory environment. CIRP/PyMT mammary glands/mammary tumors and serum had decreased cytokines that promote inflammation, angiogenesis, and metastasis compared to PyMT mammary glands and serum, documenting a shift towards an environment less supportive of tumorigenesis. CIRP overexpression also decreased CD4+ helper T cells and increased CD8+ cytotoxic T cells in mammary tumors. Overall, these data support a role for CIRP as a potent antitumor molecule that suppresses both local and systemic pro-tumorigenic inflammation.

Inhibition of anti-tumor immunity by melanoma cell-derived Activin-A depends on STING.

The transforming growth factor-β (TGF-β) family member activin A (hereafter Activin-A) is overexpressed in many cancer types, often correlating with cancer-associated cachexia and poor prognosis. Activin-A secretion by melanoma cells indirectly impedes CD8+ T cell-mediated anti-tumor immunity and promotes resistance to immunotherapies, even though Activin-A can be proinflammatory in other contexts. To identify underlying mechanisms, we here analyzed the effect of Activin-A on syngeneic grafts of Braf mutant YUMM3.3 mouse melanoma cells and on their microenvironment using single-cell RNA sequencing. We found that the Activin-A-induced immune evasion was accompanied by a proinflammatory interferon signature across multiple cell types, and that the associated increase in tumor growth depended at least in part on pernicious STING activity within the melanoma cells. Besides corroborating a role for proinflammatory signals in facilitating immune evasion, our results suggest that STING holds considerable potential as a therapeutic target to mitigate tumor-promoting Activin-A signaling at least in melanoma.

Circadian Clock Gene bmal1 Acts as a Tumor Suppressor Gene in a Mice Model of Human Glioblastoma.

Glioblastomas derived from malignant astrocytes are the most common primary tumors of the central nervous system in humans, exhibiting very bad prognosis. Treatment with surgery, radiotherapy, and chemotherapy (mainly using temozolomide), generates as much one-year survival. The circadian clock controls different aspects of tumor development, and its role in GBM is beginning to be explored. Here, the role of the canonic circadian clock gene bmal1 was studied in vivo in a nude mice model bearing human GBMs from LN229 cells xenografted orthotopically in the dorsal striatum. For that aim, a bmal1 knock-down was generated in LN229 cells by CRISPR/Cas9 gene editing tool, and tumor progression was followed in male mice by measuring survival, tumor growth, cell proliferation and prognosis with CD44 marker, as well as astrocyte activation in the tumor microenvironment with GFAP and nestin markers. Disruption of bmal1 in the tumor decreased survival, increased tumor growth and CD44 expression, worsened motor performance, as well as increased GFAP expression in astrocytes at tumor microenvironment. In addition, survival and tumor progression was not affected in mice bearing LN229 wild type GBM that underwent circadian disruption by constant light, as compared to mice synchronized to 12:12 light-dark cycles. These results consistently demonstrate in an in vivo orthotopic model of human GBM, that bmal1 has a key role as a tumor suppressor gene regulating GBM progression.

Vaccinia-related kinase 2 variants differentially affect breast cancer growth by regulating kinase activity.

Genetic information is transcribed from genomic DNA to mRNA, which is then translated into three-dimensional proteins. mRNAs can undergo various post-transcriptional modifications, including RNA editing that alters mRNA sequences, ultimately affecting protein function. In this study, RNA editing was identified at the 499th base (c.499) of human vaccinia-related kinase 2 (VRK2). This RNA editing changes the amino acid in the catalytic domain of VRK2 from isoleucine (with adenine base) to valine (with guanine base). Isoleucine-containing VRK2 has higher kinase activity than the valine-containing VRK2, which leads to an increase in tumor cell proliferation. Earlier we reported that VRK2 directly interacts with dystrobrevin-binding protein (dysbindin) and results in reducing its stability. Herein, we demonstrate that isoleucine-containing VRK2 decreases the level of dysbindin than valine-containing VRK2. Dysbindin interacts with cyclin D and thereby regulates its expression and function. The reduction in the level of dysbindin by isoleucine-containing VRK2 further enhances the cyclin D expression, resulting in increased tumor growth and reduction in survival rates. It has also been observed that in patient samples, VRK2 level was elevated in breast cancer tissue compared to normal breast tissue. Additionally, the isoleucine form of VRK2 exhibited a greater increase in breast cancer tissue. Therefore, it is concluded that VRK2, especially dependent on the 167th variant amino acid, can be one of the indexes of tumor progression and proliferation.

Breast Cancer Clinicopathology based on Neutrophil Levels at Adam Malik Hospital Medan in 2018-2021

Introduction: Breast cancer is the most frequently diagnosed cancer in the world. Breast cancer occurs due to abnormal cell growth in breast tissue so that it becomes malignant. In Indonesia, breast cancer is the most common cancer in women. That way, many markers are needed as prognostic and predictive of breast cancer. One of the prognostic factors for breast cancer is neutrophil levels combined with clinicopathological factors. Objective: Knowing the clinicopathology of breast cancer based on neutrophil levels at the Haji Adam Malik Medan General Hospital in 2018-2021. Methods: The research design is observational with a descriptive method and calculated using the Lemeshow formula. Results: Majority aged 40-49 years (39.6%), high school (44.8%), housewives (67.7%), no family history (97.9%), tumor grade II (38.5%) %), Tumor Infiltrating Lymphocytes (TILs) severe (47.9 %), mitotic score 1 (38.5 %), metastatic positive lymph node status (87.5 %), tumor size T1 (36.5 %), The number of positive and negative Estrogen Receptors is the same (50.0%), Progesterone Receptor is negative (56.3%), HER2 is 1+ (60.4%), Ki-67 is positive (84 .4 %), positive angioinvasion (63.5 %), Invasive Ductal Carcinoma (90.6 %), high neutrophil levels (77.1 %). Conclusion: High levels of neutrophils tend to result in severe Tumor Infiltrating Lymphocytes (TILs) which will increase tumor growth, occurrence of metastases, increased positive angioinvasion, negative Estrogen Receptor status, negative Progesterone Receptor status, HER2 1+ status, positive Ki-67 status.

Cancer-Associated Fibroblasts Promote Lymphatic Metastasis in Cholangiocarcinoma via the PDGF-BB/PDGFR-β Mediated Paracrine Signaling Network.

Patients with cholangiocarcinoma (CCA) with lymph node metastasis (LNM) have the worst prognosis, even after complete resection; however, the underlying mechanism remains unclear. Here, we established CAF-derived PDGF-BB as a regulator of LMN in CCA. Proteomics analysis revealed upregulation of PDGF-BB in CAFs derived from patients with CCA with LMN (LN+CAFs). Clinically, the expression of CAF-PDGF-BB correlated with poor prognosis and increased LMN in patients with CCA, while CAF-secreted PDGF-BB enhanced lymphatic endothelial cell (LEC)-mediated lymphangiogenesis and promoted the trans-LEC migration ability of tumor cells. Co-injection of LN+CAFs and cancer cells increased tumor growth and LMN in vivo. Mechanistically, CAF-derived PDGF-BB activated its receptor PDGFR-β and its downstream ERK1/2-JNK signaling pathways in LECs to promote lymphoangiogenesis, while it also upregulated the PDGFR-β-GSK-P65-mediated tumor cell migration. Finally, targeting PDGF-BB/PDGFR-β or the GSK-P65 signaling axis prohibited CAF-mediated popliteal lymphatic metastasis (PLM) in vivo. Overall, our findings revealed that CAFs promote tumor growth and LMN via a paracrine network, identifying a promising therapeutic target for patients with advanced CCA.

Primary squamous cell carcinoma of the parotid salivary gland: a clinical observation

Introduction. Malignant tumors of the salivary glands account for 3–5 % of all cases of head and neck cancer. Squamous cell carcinoma is the rarest histological variant of this pathology. It accounts for an average of 1.6 %. Among large salivary gland tumors, squamous cell carcinoma most often occurs in the parotid salivary gland.Aim. To present a clinical case of primary disseminated cancer of the parotid salivary gland with squamous cell structure.Clinical observation. A 70-year-old patient was under observation who was diagnosed with a primary squamous cell tumor of the parotid salivary gland with multiple metastases during a complex examination. He was sick for about 6 months when asymmetry of left half of the face appeared due to a painful neoplasm in the parotid salivary gland. Recently, the patient has noted increased tumor growth. Objective examination revealed peripheral paralysis of the facial nerve on the left. In the parotid-masticatory region on the left, with a transition to the zygomatic region, there is a dense painful immobile exophytic tumor with infiltration into soft tissues of the face and skin 6.5 × 5.0 cm in size with an ulcer in the center. Laterally, it reaches the tragus, and at the bottom-the angle of the lower jaw. In the submandibular region on the left, metastatic lymph nodes 2.5 × 2.0 cm in size are palpated. A biopsy was collected. Histological conclusion: non-keratinizing squamous cell carcinoma. Expression of the programmed death receptor ligand (PD-L1) in the tumor is negative. Mutations of genes Her2-neu and BRAF were not detected. According to medical imaging methods, multiple metastases were found in the lungs, liver, zygomatic bone and tumor growth in the maxillary sinus on the left. The diagnosis was established: cancer of the parotid salivary gland, stage IVC, cT4N1M1, with metastases to the liver, lungs and bones. Polychemotherapy was prescribed according to the scheme: 5-fluorouracil + cisplatin + cetuximab + bisphosphonates. After 3 cycles, a partial regression is obtained.Conclusion. The primary squamous cell carcinoma is prone to an aggressive course and has an unfavorable prognosis, especially in patients over 60 years of age with skin and facial nerve damage, as well as the presence of regional and distant metastases. Differential diagnosis is necessary to exclude the secondary nature of the tumor. The presented case is a rare example of primary squamous cell carcinoma of the parotid salivary gland with extensive metastatic lesion and regression of the pathological process after polychemotherapy in combination with targeted therapy.

Abstract C103: Non-enzymatic glycoxidation reports on society, environment and biology to inform on enduring cancer disparities

Abstract Disparities in overall cancer outcomes have been reduced across racial and ethnic divides, but still persist for some tumor types and have increased for others. This has stimulated significant debate regarding whether the enduring nature of cancer disparities arises solely out of inherent failings in addressing health inequities, or does an individual’s inherent biology also play an accompanying role. As a newly termed “bio-social determinant of health”, non-enzymatic glycoxidation profiles may represent an unexploited opportunity to consider how health inequity constructs combine to impact cancer outcomes across transdisciplinary studies. Resulting from the combined processes of glycation and oxidation, non-enzymatic glycoxidation is a complex series of reactions, leading to the irreversible posttranslational modification of peptides, lipids and nucleic acids by Advanced Glycation End Products (AGEs). Evidence is presented to support that health inequity constructs can serve to provide a superfluous source of the reactive substrates that sustains non-enzymatic glycoxidation resulting in a chronic cycle of AGE formation, bioavailability, and pathogenicity. AGEs are significant because: 1. Communities who bear the highest cancer burden are more likely to be exposed to health inequity risk factors that serve to increase AGE bioavailability 2. When health equity is compromised complex relationships are created between social and biological determinates of health that serve to overwhelm the fragile rheostat that maintains AGE homeostasis.; and 3. Increases in AGE bioavailability are associated with increased tumor growth and progression through their impact on the tumor microenvironment. Glycoxidation patterns visualized by AGE accumulation and pathogenic function may serve as a “bio-social determinant of health” as they may reflect the dynamic and varied nature of multiple health inequity constructs and their combined effects on cancer incidence and mortality. In turn this may allow a greater understanding of the complex relationships that exist between societal, ancestral, environmental, and biological constructs, and lifestyle associated risk factors associated with enduring cancer disparities. This would provide significant insight on novel cancer prevention and control strategies aimed at reducing AGE exposure due to health inequity. Citation Format: David P. Turner. Non-enzymatic glycoxidation reports on society, environment and biology to inform on enduring cancer disparities [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C103.

Abstract C074: VPS34 inhibition delays activation-induced STING degradation to prolong STING signaling and improve anti-tumor efficacy in preclinical models

Abstract Background: The STING/interferon (IFN) response pathway has been a target of cancer immunotherapy for many years, as a method for reactivating the anti-tumor immune response. Stimulation of the STING pathway leads to activation and infiltration of immune cells into the tumor, and cancer cell death. However, multiple STING agonists have failed in the clinic due to poor efficacy or toxicity1. STING activation leads to its degradation within a few hours through trafficking to the lysosome, as a mechanism to control immune cell hyperactivation2. Although this safety mechanism is important for preventing autoimmunity and chronic inflammation, it diminishes the continuous activation of immune-mediated cancer cell killing, especially in an immunosuppressive tumor microenvironment. VPS34 is a lipid kinase which plays an integral role in endosomal trafficking and autophagy3. Previous publications have demonstrated a role for VPS34 inhibition in increased effector immune cell infiltration, which synergizes with anti-PD1 therapy, demonstrating a link to immune activation4. Herein, we demonstrate that the in vivo combination efficacy with a VPS34 inhibitor and a STING agonist is due to reduced degradation of activated STING, through the inhibition of VPS34-mediated endosomal trafficking. Materials and Methods: Cancer cell lines were cultured using recommended medium. STING activity was measured by Western blot and via a CisBio pTBK1 kit. ADU-S100/MSA-2 were sourced from MedChemExpress. CCL5, CXCL10 and IFNb ELISA’s were purchased from R&D systems. Primers used to measure mRNA expression were obtained from IDT. Results: DP-9244 is a potent and selective VPS34 inhibitor. The combination of DP-9244 with the STING agonist, ADU-S100, enhances IFN-mediated gene expression and cytokine secretion in the murine melanoma cell line, B16F10, and in the human and murine myeloid cell lines THP-1 and DC2.4, demonstrating combination activity in both cancer and immune cells. This combination activity is STING-dependent as demonstrated by the complete loss of IRF and NFκB activity in the THP-1 STING-/- cell line when treated with ADU-S100, DP-9244 or the combination. Mechanistically, VPS34 inhibition delays degradation of activated STING and prolongs downstream signaling of pTBK1 in multiple human and murine cancer cell lines. Importantly, the in vitro effects extend to in vivo efficacy in the B16F10 model, in which the combination of DP-9244 and ADU-S100 results in increased tumor growth control and number of mice responding to treatment. Conclusions: These data provide a strong preclinical rationale to combine a VPS34 inhibitor with a STING agonist as a method of improving STING pathway mediated anti-tumor efficacy.

PD-1 Expression in Lymphoma Cells Mediates Cellular Proliferation By Engaging Phosphatase SHP-1/SHP-2

Introduction: Despite the improved clinical outcomes with novel therapies in patients with chronic lymphocytic leukemia (CLL), Richter transformation (RT) of CLL remains a major clinical challenge. Our group showed a selective response (~40%) with the PD-1 blocking antibody pembrolizumab in patients with RT, particularly after prior exposure to ibrutinib (Ding et al, Blood, 2017). It has been established that PD-1 inhibits cytotoxic T-cell functions via the interaction with its ligands, PD-L1 and PD-L2. However, the role of PD-1 in tumor B-cells is understudied. Here we show the expression and functional implications of the PD-1 signaling axis in B-cell pathobiology. Methods: CLL-involved lymph node (LN) (n=26) and RT (with diffuse large B-cell lymphoma [DLBCL] histology) -involved LN (n=20) samples were tested for PD-1 expression by immunohistochemistry (IHC) (mouse clone NAT105, Abcam). Then, PD-1 expression was examined in 10 lymphoma cell lines and 1 CLL-like cell line (MEC-1) by both flow cytometry (FC) and Western blot (WB) analysis. PD-1 overexpression using pLEX-lentiviral packaging system (Thermo Scientific) was evaluated for its impact on in vitro cell cycle regulation and in vivo tumor growth. CRISPR/CAS9 mediated PD-1 knockdown evaluated on cell growth in MEC-1 and Mino cells. Role of PD-1 was further examined on the regulation of cell signaling pathways using human phospho kinase array kit (R&D) and WB analysis. Additionally, gene expression signatures were evaluated by RNA-seq using pLEX-lentiviral-PD-1 transfected DLBCL cell lines OCI-LY7 and OCI-LY19. Results: PD-1 expression was significantly increased in RT-LN compared to CLL-LN (mean ± SEM in RT vs. CLL, 30.6% ± 4.7% vs. 11.5% ± 2.8%, p<0.001) as determined by IHC. PD-1 expression was highest in patients with RT where the last prior CLL therapy was ibrutinib. To test the role of PD-1 in tumor B-cells, its expression was assessed in lymphoma and CLL cell lines. The expression of PD-1 was found to be variable by both FC and WB analysis, but at very low-levels in DLBCL cell lines OCI-LY7 and OCI-LY19. Constitutive lentiviral (pLEX-PD-1)-mediated overexpression of PD-1 in OCI-LY7 and OCI-LY19 cells led to increased cell growth (1.4 and 1.9 fold, respectively, compared to original lines after 4 days of culturing of equal number of cells), which was further confirmed by cell cycle analysis which showed an increase in S phase by 20.4% (p<0.05) and 24.5% (p<0.05) in OCI-LY7 and OCI-LY19 cells, respectively. When the luciferase+ PD-1 overexpressing OCI-LY7 cells (1X10 6) were injected intravenously into NSG mice, increased tumor growth (p<0.01) was observed by bioluminescence imaging after 22 days of follow-up. PD-1 knockdown in MEC-1 and Mino cells (high basal PD-1 expression) led to significant inhibition of cell growth (85% and 79.5%, respectively, p<0.05). Using phospho-kinase array, an overall decrease of phosphorylation was detected on multiple sites of p53 (S392, S15, S46) and Chk-2 (T68) in PD-1 overexpressing OCI-LY7 and OCI-LY19 cell lines. This finding was further confirmed by WB analyses. In addition, decreased total p53 and increased phosphorylation on both SHP-1 and SHP-2 were found in PD-1 overexpressing OCI-LY7 and OCI-LY19 cell lines. SHP-1 (TPI-1) and SHP-1+SHP-2 (TPI-1+TNO155) inhibitor treatment promoted OCI-LY19 cell death and led to recovery of phosphorylation on ATM, Chk-2 and p53 in these cells. RNA sequencing data in PD-1 overexpressing OCI-LY7 and OCI-LY19 cells revealed increased expression of genes that promote cell cycle progression and DNA replication. Conclusion: Our data showed robust PD-1 expression in patients with Richter transformation from CLL to DLBCL. PD-1 overexpression in DLBCL lymphoma cell lines enhanced cell proliferation in vitro and in vivo. Further investigation identified that PD-1 modified the phosphorylation/function of SHP phosphatases and thereby regulated p53 pathways ( Figure 1). These findings indicate that RT with DLBCL cells have the ability to “hijack” the PD-1 pathway which results in downregulation of p53 mediated DNA repair. This novel finding provides for new strategies to further evaluate the interaction of PD-1 signals with DNA repair pathways and suggest potential novel targets for RT.

PA suppresses antitumor immunity of T cells by disturbing mitochondrial activity through Akt/mTOR-mediated Ca2+ flux

Deciphering the mechanisms behind how T cells become exhausted and regulatory T cells (Tregs) differentiate in a tumor microenvironment (TME) will significantly benefit cancer immunotherapy. A common metabolic alteration feature in TME is lipid accumulation, associated with T cell exhaustion and Treg differentiation. However, the regulatory role of free fatty acids (FFA) on T cell antitumor immunity has yet to be clearly illustrated. Our study observed that palmitic acid (PA), the most abundant saturated FFA in mouse plasma, enhanced T cell exhaustion and Tregs population in TME and increased tumor growth. In contrast, oleic acid (OA), a monounsaturated FFA, rescued PA-induced T cell exhaustion, decreased Treg population, and ameliorated T cell antitumor immunity in an obese mouse model. Mechanistically, mitochondrial metabolic activity is critical in maintaining T cell function, which PA attenuated. PA-induced T cell exhaustion and Treg formation depended on CD36 and Akt/mTOR-mediated calcium signaling. The study described a new mechanism of PA-induced downregulation of antitumor immunity of T cells and the therapeutic potential behind its restoration by targeting PA.

A bedside to bench study of anti-PD-1, anti-CD40, and anti-CSF1R indicates that more is not necessarily better

BackgroundStimulating inflammatory tumor associated macrophages can overcome resistance to PD-(L)1 blockade. We previously conducted a phase I trial of cabiralizumab (anti-CSF1R), sotigalimab (CD40-agonist) and nivolumab. Our current purpose was to study the activity and cellular effects of this three-drug regimen in anti-PD-1-resistant melanoma.MethodsWe employed a Simon’s two-stage design and analyzed circulating immune cells from patients treated with this regimen for treatment-related changes. We assessed various dose levels of anti-CSF1R in murine melanoma models and studied the cellular and molecular effects.ResultsThirteen patients were enrolled in the first stage. We observed one (7.7%) confirmed and one (7.7%) unconfirmed partial response, 5 patients had stable disease (38.5%) and 6 disease progression (42.6%). We elected not to proceed to the second stage. CyTOF analysis revealed a reduction in non-classical monocytes. Patients with prolonged stable disease or partial response who remained on study for longer had increased markers of antigen presentation after treatment compared to patients whose disease progressed rapidly. In a murine model, higher anti-CSF1R doses resulted in increased tumor growth and worse survival. Using single-cell RNA-sequencing, we identified a suppressive monocyte/macrophage population in murine tumors exposed to higher doses.ConclusionsHigher anti-CSF1R doses are inferior to lower doses in a preclinical model, inducing a suppressive macrophage population, and potentially explaining the disappointing results observed in patients. While it is impossible to directly infer human doses from murine studies, careful intra-species evaluation can provide important insight. Cabiralizumab dose optimization is necessary for this patient population with limited treatment options.Trial registrationClinicalTrials.gov Identifier: NCT03502330.

DNAR-09. THE ROLE OF MSH2 IN GLIOBLASTOMA GROWTH AND TEMOZOLOMIDE RESISTANCE

Abstract Despite all the advances in understanding and finding new therapeutic approaches, glioblastoma (GBM) remains incurable. The standard of care is surgical resection when feasible, combined with radiotherapy and chemotherapy using temozolomide (TMZ). Different mechanisms have been suggested to contribute to this failure: intrinsic or acquired resistance to TMZ and deficiencies in DNA mismatch repair (MMR) genes such as MSH2 that promote tumor progression. In this study, we have identified a correlation between low MSH2 expression and shorter patient survival utilizing patient GBM samples from the TCGA. Furthermore, we have generated immunocompetent genetically engineered mouse models (GEMMs) of GBM with germline or somatic loss of Msh2 to test the biological significance of Msh2 in GBM growth and resistance to TMZ and immunotherapy. We have generated tumors with reduced or abrogated Msh2 using GEMMs based on the RCAS/tv-a gene transfer system in combination with mice that exhibited heterozygous and homozygous loss of Msh2. Tumors with overexpression of PDGFB in combination with Tp53 silencing in mice with reduced or deficient Msh2 demonstrated shorter survival time and increased tumor growth compared to wild-type tumor-bearing mice. In addition, our results indicate that Msh2 deficiency increases the malignancy of gliomas. Finally, our data demonstrate that TMZ treatment for two weeks provides a significant survival advantage in WT tumor-bearing mice, while no efficacy was observed in tumor-bearing mice with either heterozygous or homozygous loss of Msh2, recapitulating what is observed in clinics. We are currently evaluating the role of Msh2 in the efficacy of immune checkpoint inhibitors in GBM.

Cathepsin L-mediated EGFR cleavage affects intracellular signalling pathways in cancer.

Proteolytic activity in the tumour microenvironment is an important factor in cancer development since it can also affect intracellular signalling pathways via positive feedback loops that result in either increased tumour growth or resistance to anticancer mechanisms. In this study, we demonstrated extracellular cathepsin L-mediated cleavage of epidermal growth factor receptor (EGFR) and identified the cleavage site in the extracellular domain after R224. To further evaluate the relevance of this cleavage, we cloned and expressed a truncated version of EGFR, starting at G225, in HeLa cells. We confirmed the constitutive activation of the truncated protein in the absence of ligand binding and determined possible changes in intracellular signalling. Furthermore, we determined the effect of truncated EGFR protein expression on HeLa cell viability and response to the EGFR inhibitors, tyrosine kinase inhibitor (TKI) erlotinib and monoclonal antibody (mAb) cetuximab. Our data reveal the nuclear localization and phosphorylation of EGFR and signal trancducer and activator of transcription 3 (STAT3) in cells that express the truncated EGFR protein and suggest that these phenomena cause resistance to EGFR inhibitors.

OR16-02 Histone ADP-Ribosylation Site Mutations As Oncogenic Drivers In Breast And Ovarian Cancers

Abstract Disclosure: D. Huang: None. C.V. Camacho: None. A. Nagari: None. W.L. Kraus: Other; Self; - Founder and stockholder for Ribon Therapeutics, Inc. - Founder, stockholder, scientific advisory board member, member of the board of directors for ARase Therapeutics, Inc. - Coholder of U.S. Patent. Recent studies have identified oncohistones, which are encoded by histone genes that harbor missense mutations, as a new class of cancer drivers. We found that many of the oncohistone mutations identified with the highest frequencies in human cancers occur on glutamate (Glu, E) and aspartate (Asp, D) residues, which are potential sites of ADP-ribosylation (ADPRylation). ADPRylation is a post-translational modification, which is catalyzed by the PARP family of ADP-ribosyl transferases and results in the covalent attachment of ADP-ribose on protein substrates. Unlike other well characterized histone modifications, the functions of histone ADPRylation are poorly characterized. Mass spectrometry analyses confirmed that 20 Glu or Asp residues subjected to oncohistone mutation are bona fide sites of ADPRylation by PARP-1. Ectopic expression of each of six different oncohistone mutants from three different core histones (H2B, H3, and H4) altered the proliferation of at least 2 out of 3 cancer cell lines, including MCF-7 and MDA-MB-231 breast cancer cells, as well as OVCAR-3 ovarian cancer cells. We focused on H2B-D51, whose mutation resulted in enhanced cell proliferation in both of the breast cancer cell lines for further characterization of its potential transcription regulatory effects. Proteomic (histone PTMs) and genomic (ATAC-Seq, ChIP-Seq and RNA-Seq) analyses revealed that loss of ADPRylation on H2B-D51 by mutation to alanine (Ala, A) or asparagine (Asn, N) dramatically enhanced p300-mediated acetylation of H2B, altered chromatin accessibility, and changed the pattern of gene expression in MDA-MB-231 cells. To determine the effects of H2B-D51 ADPRylation on cancer cell growth in vivo and their responses to the FDA-approved PARP inhibitor Niraparib, we performed xenograft tumor growth assays in immunocompromised mice using MDA-MB-231 cells ectopically expressing wild-type or D51A mutant histone H2B. Ectopic expression of the H2B-D51A mutant led to a significant increase in tumor growth versus wild-type H2B. Interestingly, these effects were inhibited by treatment with Niraparib, suggesting that the H2B-D51A mutant alone does not confer resistance to PARP inhibitor. Taken together, this study demonstrates that functional sites of Glu/Asp ADPRylation on histones are mutated in cancers, allowing cancer cells to escape the growth-regulating effects of ADPRylation. (This work was supported by grants from the NIH/NIDDK and CPRIT, and funds from the Cecil H. and Ida Green Center for Reproductive Biology Sciences Endowment to W.L.K.) Presentation: Friday, June 16, 2023

Rigosertib promotes anti-tumor immunity via autophagic degradation of PD-L1 in colorectal cancer cells

Rigosertib (RGS) is a benzyl styryl sulfone which exhibits impressive cytotoxicity in cancer cells. However, its modulating effect on tumor immune microenvironment remains elusive. In our experiments, compared with immunodeficient mouse model, increased tumor growth arrest and robust anti-tumor immunity were observed in RGS-treated colorectal cancer (CRC) isograft tumors in immunocompetent mice. Intriguingly, RGS markedly down-regulated programmed cell death ligand 1 (PD-L1) expression in both vivo and in vitro. Meanwhile, RGS increased autophagic vacuole number in CRC cells as seen by transmission electron microscopy and immunofluorescence. Moreover, increased LC3-II level and tandem-mRFP- GFP- LC3 labeled vacuole accumulation demonstrated RGS-induced autophagic flux. Mechanistically, it is the activation of AMP-activated protein kinase-UNC-51-like kinase 1 (AMPK-ULK1) axis, rather than the canonical mTOR signaling pathway, that plays a pivotal role in RGS-induced autophagy. AMPK-ULK1 dependent autophagy inhibition, by either short interfering RNA or chemical inhibitors, blocked RGS-induced PD-L1 degradation. Finally, RGS exhibited synergistic anti-tumor activity with cytotoxic T-lymphocyte-associated protein 4 monoclonal antibody in the CRC isograft model. Furthermore, apart from the immunomodulatory effect, we also confirmed the direct cytotoxicity of RGS in inducing mitochondria-related apoptosis. Altogether, considering its PD-L1 inhibitory and cytotoxic effects, RGS could be a promising drug for CRC therapy.

OR05-01 Myeloid Cell Intrinsic NR0B2 Inhibits Regulatory T Cell Expansion Within The Tumor Microenvironment: Potential Novel Strategy For Breast Cancer Immunotherapy

Abstract Disclosure: H. Vidana Gamage: None. S. Shahoei: None. S. Albright: None. T. Nguyen: None. R. Farmer: None. E. Weisser: None. R. Bautista Rivas: None. C.P. Schane: None. Y. Wang: None. A. Nelczyk: None. L. Ma: None. S. Tiwari: None. A. Das Gupta: None. S. Bendre: None. L. Apetoh: None. P. Hergenrother: None. E.R. Nelson: None. Breast cancer remains the second leading cause of cancer-related death in women. Immune-oncology interventions for the treatment of breast cancer have thus far been underwhelming, providing strong rationale for the development of drugs to increase their efficacy. A major obstacle to immunotherapy is the highly immunosuppressive tumor microenvironment. This phenomenon is strongly maintained by myeloid cells and regulatory T cells (Tregs), which are associated with immune escape, poor prognosis and treatment resistance. Importantly, myeloid cells are also critical for a robust anti-tumor response. Therefore, we require strategies to re-educate myeloid cells rather than eliminating them. Elevated cholesterol is a risk factor for breast cancer, while cholesterol-lowering drugs are associated with a good prognosis. Previous work has demonstrated that certain downstream metabolites of cholesterol, can promote immune escape and metastatic progression. The nuclear receptor NR0B2 is a critical negative regulator of cholesterol homeostasis. Mining clinical data, we have found that high tumoral expression of NR0B2 is associated with an increased time to recurrence. Single cell RNA-sequencing indicates that NR0B2 is expressed within the myeloid cells of normal breast tissue. Thus, we hypothesized that myeloid cell NR0B2 is involved in breast cancer pathophysiology and can be used as a druggable target. Intriguingly, overexpression of NR0B2 or activation with a small molecule agonist in murine bone marrow derived macrophages (BMDMs) or dendritic cells skewed T cell expansion away from Tregs. Conversely, Treg expansion increased when NR0B2 was knocked down. In order to investigate the role of NR0B2 in vivo, we monitored the growth of tumors grafted into mice that had NR0B2 genetically knocked out in the myeloid lineage (NR0B2fl/fl;LysMCre+) and respective control mice (NR0B2wt/wt;LysMCre+). Mice lacking NR0B2 exhibited significantly increased tumor growth and elevated tumor infiltrating Tregs. Additionally, human breast tumors samples indicated an inverse correlation between NR0B2 and FOXP3 (marker for Tregs). We further investigated the downstream targets of NR0B2 mediating this anti-tumor phenotype and identified that NLRP3 inflammasome-IL1β axis is a likely modulator in re-educating myeloid cell-Treg function. Importantly, a putative small molecule agonist decreased established metastatic lesions and increased the efficacy of αPD-L1 therapy. Subsequent medicinal chemistry was used to develop a novel NR0B2 agonist with strong anti-metastatic properties when used as a single agent in a preclinical mouse model. Collectively, our data implicates NR0B2 within myeloid cells as a modulator of Tregs, a cell population that has thus far been therapeutically intractable. Therefore, NR0B2 may prove to be a promising therapeutic strategy to improve breast cancer immunotherapy. Presentation: Thursday, June 15, 2023