Combination Treatment Targeting Research Articles

Shifting Mycobacterial Serine Hydrolase Activity Visualized Using Multi-Layer In-Gel Activity Assays.

The ability of Mycobacterium tuberculosis to derive lipids from the host, store them intracellularly, and then break them down into energy requires a battery of serine hydrolases. Serine hydrolases are a large, diverse enzyme family with functional roles in dormant, active, and reactivating mycobacterial cultures. To rapidly measure substrate-dependent shifts in mycobacterial serine hydrolase activity, we combined a robust mycobacterial growth system of nitrogen limitation and variable carbon availability with nimble in-gel fluorogenic enzyme measurements. Using this methodology, we rapidly analyzed a range of ester substrates, identified multiple hydrolases concurrently, observed functional enzyme shifts, and measured global substrate preferences. Within every growth condition, mycobacterial hydrolases displayed the full, dynamic range of upregulated, downregulated, and constitutively active hydrolases independent of the ester substrate. Increasing the alkyl chain length of the ester substrate also allowed visualization of distinct hydrolase activity likely corresponding with lipases most responsible for lipid breakdown. The most robust expression of hydrolase activity was observed under the highest stress growth conditions, reflecting the induction of multiple metabolic pathways scavenging for energy to survive under this high stress. The unique hydrolases present under these high-stress conditions could represent novel drug targets for combination treatment with current front-line therapeutics. Combining diverse fluorogenic esters with in-gel activity measurements provides a rapid, customizable, and sensitive detection method for mycobacterial serine hydrolase activity.

DDDR-11. IDENTIFYING NEW VULNERABILITIES TO TARGET PEDIATRIC LOW-GRADE GLIOMA

Abstract Pediatric low-grade gliomas (pLGGs) are the most common solid tumors in children and are associated with devastating lifelong morbidities and mortality. Recent genomic profiling efforts have revealed that these tumors are largely driven by single-driver events that activate MAPK signaling. These insights have led to early clinical trials evaluating the role of MAPK inhibitors for these children with promising initial results. However, pLGGs are not cured by MAPK inhibitors, and tumors often rapidly rebound upon cessation of treatment. Therefore, continuous dosing of these inhibitors is required throughout a child’s development–often with significant and potentially permanent toxicities. In addition, 30-40% of tumors do not respond to MAPK inhibition, indicating primary resistance. We hypothesized that pediatrlc low-grade gliomas may also harbor additional dependencies beyond the MAPK pathway that may represent potential therapeutic targets. To address this, we generated isogenic neural stem cell models that expressed pLGG relevant oncogenes (BRAF family members, FGFR family members and MYB transcription factors, and subjected them to genome-scale CRISPR-cas9 screens. Compared to vector controls, pLGG models exhibited genetic dependencies on pathways that included regulators of cell-cycle and differentiation, in addition to genes involved in regulating the mTOR and MAPK pathways. Cell lines expressing MYB family transcription factors also harbored dependencies on genes involved in the DREAM complex. Together, these genes and pathways represent potential targets for combination treatments with MAPK pathway inhibitors for the treatment of pediatric low-grade gliomas.

Metastatic Castration-Resistant Prostate Cancer, Immune Checkpoint Inhibitors, and Beyond.

The therapeutic landscape of several genitourinary malignancies has been revolutionized by the development of immune checkpoint inhibitors (ICIs); however, the utility of immunotherapies in prostate cancer has been limited, partly due to the immunologically "cold" tumor terrain of prostate cancer. As of today, pembrolizumab is the only immune checkpoint inhibitor approved for the treatment of metastatic castration resistant prostate cancer (mCRPC) in a select group of patients with high microsatellite instability (MSI-H), deficient mismatch repair (dMMR), or high tumor mutational burden (TMB). Looking ahead, several combinatorial approaches with ICIs involving radioligands, radiotherapy, PARP inhibitors, interleukin inhibitors, and cancer vaccines are exploring a potential synergistic effect. Furthermore, B7-H3 is an alternative checkpoint that may hold promise in adding to the treatment landscape of mCRPC. This review aims to summarize previous monotherapy and combination therapy trials of ICIs as well as novel immunotherapy combination therapeutic strategies and treatment targets in mCRPC.

Combination Treatment Targeting mTOR and MAPK Pathways Has Synergistic Activity in Multiple Myeloma

Multiple myeloma (MM) is an incurable, malignant B cell disorder characterized by frequent relapses and a poor prognosis. Thus, new therapeutic approaches are warranted. The phosphatidylinositol-3-kinase (PI3K) pathway plays a key role in many critical cellular processes, including cell proliferation and survival. Activated PI3K/AKT (protein kinases B)/mTOR (mammalian target of rapamycin) signaling has been identified in MM primary patient samples and cell lines. In this study, the efficacy of PI3K and mTOR inhibitors in various MM cell lines representing three different prognostic subtypes was tested. Whereas MM cell lines were rather resistant to PI3K inhibition, treatment with the mTOR inhibitor temsirolimus decreases the phosphorylation of key molecules in the PI3K pathway in MM cell lines, leading to G0/G1 cell cycle arrest and thus reduced proliferation. Strikingly, the efficacy of temsirolimus was amplified by combining the treatment with the Mitogen-activated protein kinase kinase (MEK) inhibitor trametinib. Our findings provide a scientific rationale for the simultaneous inhibition of mTOR and MEK as a novel strategy for the treatment of MM.

Targeting the Heterogeneous Genomic Landscape in Triple-Negative Breast Cancer through Inhibitors of the Transcriptional Machinery.

Simple SummaryTriple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with limited options for therapy. Cancer development and maintenance is dependent on the production of oncogenic proteins. In TNBC, the production of many of these proteins is changed, and therapeutic targeting of one of these proteins is often not effective. However, a common step in the production of these proteins, transcription, can be effectively targeted through inhibition of the transcriptional machinery. These inhibitors can specifically suppress the production of oncogenic proteins important for TNBC. At the same time, the altered production of these proteins might interfere with the sensitivity of these cancer cells to these inhibitors. This review provides an overview of the effects of inhibitors of the transcriptional machinery on the abnormal oncogenic protein production in TNBC. This overview thereby highlights the research further needed in this field, and the potential opportunities of using these inhibitors for TNBC.Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer defined by lack of the estrogen, progesterone and human epidermal growth factor receptor 2. Although TNBC tumors contain a wide variety of oncogenic mutations and copy number alterations, the direct targeting of these alterations has failed to substantially improve therapeutic efficacy. This efficacy is strongly limited by interpatient and intratumor heterogeneity, and thereby a lack in uniformity of targetable drivers. Most of these genetic abnormalities eventually drive specific transcriptional programs, which may be a general underlying vulnerability. Currently, there are multiple selective inhibitors, which target the transcriptional machinery through transcriptional cyclin-dependent kinases (CDKs) 7, 8, 9, 12 and 13 and bromodomain extra-terminal motif (BET) proteins, including BRD4. In this review, we discuss how inhibitors of the transcriptional machinery can effectively target genetic abnormalities in TNBC, and how these abnormalities can influence sensitivity to these inhibitors. These inhibitors target the genomic landscape in TNBC by specifically suppressing MYC-driven transcription, inducing further DNA damage, improving anti-cancer immunity, and preventing drug resistance against MAPK and PI3K-targeted therapies. Because the transcriptional machinery enables transcription and propagation of multiple cancer drivers, it may be a promising target for (combination) treatment, especially of heterogeneous malignancies, including TNBC.

Targeting of the Lipid Metabolism Impairs Resistance to BRAF Kinase Inhibitor in Melanoma.

Drug resistance limits the achievement of persistent cures for the treatment of melanoma, in spite of the efficacy of the available drugs. The aim of the present study was to explore the involvement of lipid metabolism in melanoma resistance and assess the effects of its targeting in cellular models of melanoma with acquired resistance to the BRAF-inhibitor PLX4032/Vemurafenib. Since transcriptional profiles pointed to decreased cholesterol and fatty acids synthesis in resistant cells as compared to their parental counterparts, we examined lipid composition profiles of resistant cells, studied cell growth dependence on extracellular lipids, assessed the modulation of enzymes controlling the main nodes in lipid biosynthesis, and evaluated the effects of targeting Acetyl-CoA Acetyltransferase 2 (ACAT2), the first enzyme in the cholesterol synthesis pathway, and Acyl-CoA Cholesterol Acyl Transferase (ACAT/SOAT), which catalyzes the intracellular esterification of cholesterol and the formation of cholesteryl esters. We found a different lipid composition in the resistant cells, which displayed reduced saturated fatty acids (SFA), increased monounsaturated (MUFA) and polyunsaturated (PUFA), and reduced cholesteryl esters (CE) and triglycerides (TG), along with modulated expression of enzymes regulating biosynthetic nodes of the lipid metabolism. The effect of tackling lipid metabolism pathways in resistant cells was evidenced by lipid starvation, which reduced cell growth, increased sensitivity to the BRAF-inhibitor PLX4032, and induced the expression of enzymes involved in fatty acid and cholesterol metabolism. Molecular targeting of ACAT2 or pharmacological inhibition of SOAT by avasimibe showed antiproliferative effects in melanoma cell lines and a synergistic drug interaction with PLX4032, an effect associated to increased ferroptosis. Overall, our findings reveal that lipid metabolism affects melanoma sensitivity to BRAF inhibitors and that extracellular lipid availability may influence tumor cell response to treatment, a relevant finding in the frame of personalized therapy. In addition, our results indicate new candidate targets for drug combination treatments.

IGFBP7 and the Tumor Immune Landscape: A Novel Target for Immunotherapy in Bladder Cancer.

Insulin-like growth factor binding protein-7 (IGFBP7) was recently reported to be a ligand of CD93, a potential target to normalize vasculature and attenuate immunotherapy. However, its role in the tumor microenvironment (TME) and immunotherapy response of bladder cancer (BLCA) remains unclear. We comprehensively evaluated the correlation between IGFBP7 and multiple immunological characteristics of BLCA across The Cancer Genome Atlas (TCGA) and two external cohorts. Importantly, the response of IGFBP7-grouped BLCA patients to immunotherapy was predicted and validated by five real-word immunotherapy cohorts. Finally, we developed an IGFBP7-based immune risk model validated by five independent cohorts. IGFBP7 modulated the TME across pan-caners. In BLCA, high expression of IGFBP7 was correlated with more aggressive clinical features. IGFBP7 was positively associated with immunomodulators and promoted tumor-infiltrating lymphocyte trafficking into the tumor microenvironment. However, T cells recognition and tumor cell killing were lower in the high-IGFBP7 group. In addition, high expression of IGFBP7 displayed lower enrichment scores for most pro-immunotherapy pathways. Clinical data from IMvigor210 and GSE176307 indicated that IGFBP7 negatively correlated with the BLCA immunotherapy response. The same trend was also observed in a renal cell carcinoma (RCC) cohort and two melanoma cohorts. Notably, urothelial and luminal differentiation were less frequently observed in the high-IGFBP7 group, while neuroendocrine differentiation was more frequently observed. Mechanistically, high IGFBP7 was associated with an enriched hypoxia pathway and higher expression of key genes in ERBB therapy and antiangiogenic therapy. Furthermore, our IGFBP7-based immune risk model was able to predict the prognosis and response to immunotherapy with good accuracy (5-year AUC = 0.734). Overall, IGFBP7 plays a critical role in the immunoregulation and TME of BLCA and may serve as a novel potential target for combination treatment with immunotherapy for BLCA.

Abstract 3281: DNMT targeting enhances vulnerability of glioblastoma cells to MNK inhibition

Abstract Many factors complicate therapeutic strategies for glioblastoma (GBM), including the existence of the blood brain barrier and a heterogenous population of difficult to treat glioma stem cells. Innovative strategies targeting novel pathways alone or in combination are needed for sustainable therapeutic improvements. The MAPK pathway has been implicated in many cancers. MAPK interacting kinases (MNK1 and MNK2) are downstream of MAPKs and phosphorylate the eukaryotic translation initiation factor 4E (eIF4E), a protein involved in translation of oncogenic mRNAs. We have previously established pharmacological MNK inhibition as a promising strategy for GBM. However, most currently available MNK inhibitors lack specificity and exhibit off-target effects. We developed novel selective MNK inhibitors that show MNK inhibition specificity in GBM established cell lines as well as patient-derived cell lines propagated under stem cell permissive conditions as 3-D neurospheres. MNK inhibitors reduced cell viability and neurosphere growth. Our previous work with MNK inhibitors showed involvement in negative feedback loops activated with treatment of other pharmacological agents, so we conducted a high-throughput screening to identify potential targets for combination treatment. One of the top hits was a DNA methyltransferase (DNMT) inhibitor that enhanced MNK inhibitor antineoplastic effects in GBM cells. Dual MNK and DNMT inhibition synergistically reduced neurosphere growth in 3-D glioma stem-like cells. The combination promoted apoptosis in the mesenchymal glioma stem-like cells as shown through flow cytometry and increased expression of cleaved PARP, cleaved caspase 3, and Bax. Also, DNMT targeting enhanced the viability reduction effects of siRNA mediated MNK1 knockdown in GBM cells. This combination of our novel MNK inhibitor with DNMT inhibition elicited antineoplastic benefits in both 2-D cultures and 3-D glioma stem cell-like populations, demonstrating a potential novel therapeutic strategy in GBM. Citation Format: Candice Mazewski, Frank Eckerdt, Aneta Baran, Mariafausta Fischietti, Purav P. Vagadia, Ricardo E. Perez, Charles D. James, Gary E. Schiltz, Leonidas C. Platanias. DNMT targeting enhances vulnerability of glioblastoma cells to MNK inhibition [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 3281.

Abstract 1857: Glioblastoma growth is suppressed dual inhibition of EGFR and CDK6 kinases

Abstract Glioblastoma (GBM) is a malignant brain tumor that has proven difficult to treat, despite expressing promising targets such as EGFRvIII. EGFRvIII, a mutant version of the epidermal growth factor receptor (EGFR), is constitutively active and not present in normal brain cells. The tumor specificity of EGFRvIII and the frequent EGFR amplification seen in GBM make EGFR a potentially attractive therapeutic target; however, clinical studies have shown little to no efficacy for EGFR tyrosine kinase inhibitors (TKI). One reason for this lack of efficacy may be adaptive resistance. We used RNA sequencing and multiplexed inhibitor beads with mass spectrometry (MIB-MS) to study the transcriptomes and kinomes of genetically engineered mouse astrocytes to investigate this resistance and identify potential targets for dual inhibition. Out of 329 kinases detected by MIB-MS, 76 were differentially expressed between cells with Cdkn2a deletion (“C”) and cells that also overexpressed EGFRvIII (“CEv3”). Thirty-four of these kinases were overexpressed in the CEv3 cells relative to the parental C cells (log2 fold change of 5.6, p<1x105). One of these kinases, Cdk6, is also significantly overexpressed in CEv3 cells versus cells that have a further loss of function mutation of Pten (“CEv3P”) (log2 fold change of 5.6, p<1x105). Despite this significant differential expression at the protein level, RNA expression of Cdk6 was similar between cell lines. When these cells were treated with the CDK6 inhibitor abemaciclib, CEv3 cells were found to be significantly more sensitive to inhibition than C and CEv3P cells (IC50 of 0.10 μM vs. 0.18 μM and 0.23 μM, respectively). Similarly, when cells were treated with abemaciclib in combination with the EGFR inhibitor neratinib, there was significantly higher synergy in CEv3 cells than C or CEv3P cells. Genotypically-matched patient-derived xenograft (PDX) cells were assayed for EGFR-CDK6 inhibitor synergy and showed a similar pattern of greater synergy in cells with EGFRvIII overexpression and functional PTEN than cells with EGFRvIII overexpression and PTEN loss. CEv3 and CEv3P cells were orthotopically implanted into mice and treated with neratinib, abemaciclib, or a combination. In CEv3-injected mice, combination treatment led to significantly longer survival than either single agent or control treatment. However, in CEv3P-injected mice, no survival difference was seen between any of the treatment arms. Taken together, these data provide strong evidence that CDK6 is a promising target for combination treatment with EGFR inhibitors in glioblastoma. Citation Format: Erin Smithberger, Abigail K. Shelton, Ryan E. Bash, Madison K. Butler, Alex R. Flores, Allie Stamper, Steven P. Angus, Michael P. East, Gary L. Johnson, Michael E. Berens, Frank B. Furnari, Ryan Miller. Glioblastoma growth is suppressed dual inhibition of EGFR and CDK6 kinases [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 1857.

Inhibition of Core Binding Factor Transcriptional Complex Provides a Novel Pharmacological Target for Osteosarcoma

Osteosarcoma (OS) is the most common primary malignant bone tumor in canines and humans. However, there have been no new successful therapies that improve long‐term survival in several decades. This indicates the need for novel therapeutic targets for treating OS. Core binding factor is a heterodimeric transcriptional complex, comprised of proteins runt‐related transcription factor 2 (RUNX2) and core‐binding factor subunit Beta (CBFβ), that promotes transcription of genes related to bone formation, osteoblast differentiation, and bone mineralization. RUNX2 protein expression is deregulated in OS tumors which contributes to chemoresistance, increased proliferation, and defective differentiation, suggesting it could be a therapeutic target in OS. This study utilizes small molecule allosteric inhibitor of CBFβ, AI‐14‐91, to target the interaction of RUNX2 and CBFβ, thereby reducing RUNX2 binding to target genes, in order to evaluate the CBFβ and RUNX2 interaction as a pharmacological target in OS. We hypothesized that AI‐14‐91 will reduce the malignant phenotype of human OS cell lines by disrupting the RUNX2‐CBFβ interaction.In vitro assays with compound AI‐14‐91 were performed with a panel of unique human OS cell lines (LM7, MG‐63, Saos‐2, SJSA‐1, and U2OS). This panel contains unique genetic mutations in key tumor‐related genes such as p53, MDM2, Rb1, or CDKN2A. RUNX2 knockout (KO) and CBFβ KO cell lines were also generated from the parental U2OS by CRISPR/Cas9 editing, which was confirmed via sequencing and western blot. Concentration‐ and time‐dependent proliferation assays were performed with all cell lines, demonstrating AI‐14‐91 IC50 values ranging from 13‐25 µM across the panel. Clonogenic assays showed AI‐14‐91 treatment significantly reduced colony formation in the majority of cell lines. Flow cytometry revealed cell cycle progression was altered in the parental U2OS and the RUNX2 KO and CBFβ KO cell lines with a significant decrease of cell number in G0/G1 phase and an increase in S phase upon 48 exposure to 20 µM AI‐14‐91. Apoptotic and necrotic cell populations in parental U2OS and KO lines were quantified by flow cytometry bivariate analysis with Annexin V/PI co‐stain. Batch 3’ Tag‐Seq RNAseq analysis was performed on parental U2OS cells exposed to 5 or 20 µM AI‐14‐91 for 6 or 48 hours and on RUNX2 KO and CBFβ KO cell lines. Differential gene expression (DGE) and enrichment analysis was performed to evaluate pertinent transcriptional pathways altered by inhibitor treatment, and to compare the effects of inhibitor treatment with knockout of putative inhibitor targets.Treatment with AI‐14‐91 has shown desirable antitumor effects in human OS cell lines that may be independent of the absence of proteins RUNX2 or CBFβ. Further analysis into DGE pathways combined with ChIP assays will help delineate mechanisms of action and identify additional potential targets for combination treatments. Orthotopic mouse models utilizing the metastasizing LM7 cell line with provide further information on the potential clinical utility of targeting the RUNX2‐CBFβ interaction in OS.

Tumor Drug Concentration and Phosphoproteomic Profiles After Two Weeks of Treatment With Sunitinib in Patients with Newly Diagnosed Glioblastoma.

Tyrosine kinase inhibitors (TKI) have poor efficacy in patients with glioblastoma (GBM). Here, we studied whether this is predominantly due to restricted blood-brain barrier penetration or more to biological characteristics of GBM. Tumor drug concentrations of the TKI sunitinib after 2 weeks of preoperative treatment was determined in 5 patients with GBM and compared with its in vitro inhibitory concentration (IC50) in GBM cell lines. In addition, phosphotyrosine (pTyr)-directed mass spectrometry (MS)-based proteomics was performed to evaluate sunitinib-treated versus control GBM tumors. The median tumor sunitinib concentration of 1.9 μmol/L (range 1.0-3.4) was 10-fold higher than in concurrent plasma, but three times lower than sunitinib IC50s in GBM cell lines (median 5.4 μmol/L, 3.0-8.5; P = 0.01). pTyr-phosphoproteomic profiles of tumor samples from 4 sunitinib-treated versus 7 control patients revealed 108 significantly up- and 23 downregulated (P < 0.05) phosphopeptides for sunitinib treatment, resulting in an EGFR-centered signaling network. Outlier analysis of kinase activities as a potential strategy to identify drug targets in individual tumors identified nine kinases, including MAPK10 and INSR/IGF1R. Achieved tumor sunitinib concentrations in patients with GBM are higher than in plasma, but lower than reported for other tumor types and insufficient to significantly inhibit tumor cell growth in vitro. Therefore, alternative TKI dosing to increase intratumoral sunitinib concentrations might improve clinical benefit for patients with GBM. In parallel, a complex profile of kinase activity in GBM was found, supporting the potential of (phospho)proteomic analysis for the identification of targets for (combination) treatment.

Synergistic activity of combined inhibition of anti-apoptotic molecules in B-cell precursor ALL

Targeting BCL-2, a key regulator of survival in B-cell malignancies including precursor B-cell acute lymphoblastic leukemia, has become a promising treatment strategy. However, given the redundancy of anti-apoptotic BCL-2 family proteins (BCL-2, BCL-XL, MCL-1), single targeting may not be sufficient. When analyzing the effects of BH3-mimetics selectively targeting BCL-XL and MCL-1 alone or in combination with the BCL-2 inhibitor venetoclax, heterogeneous sensitivity to either of these inhibitors was found in ALL cell lines and in patient-derived xenografts. Interestingly, some venetoclax-resistant leukemias were sensitive to the MCL-1-selective antagonist S63845 and/or BCL-XL-selective A-1331852 suggesting functional mutual substitution. Consequently, co-inhibition of BCL-2 and MCL-1 or BCL-XL resulted in synergistic apoptosis induction. Functional analysis by BH3-profiling and analysis of protein complexes revealed that venetoclax-treated ALL cells are dependent on MCL-1 and BCL-XL, indicating that MCL-1 or BCL-XL provide an Achilles heel in BCL-2-inhibited cells. The effect of combining BCL-2 and MCL-1 inhibition by venetoclax and S63845 was evaluated in vivo and strongly enhanced anti-leukemia activity was found in a pre-clinical patient-derived xenograft model. Our study offers in-depth molecular analysis of mutual substitution of BCL-2 family proteins in acute lymphoblastic leukemia and provides targets for combination treatment in vivo and in ongoing clinical studies.

In Vivo Screen Identifies Liver X Receptor Alpha Agonism Potentiates Sorafenib Killing of Hepatocellular Carcinoma.

In Vivo Screen Identifies Liver X Receptor Alpha Agonism Potentiates Sorafenib Killing of Hepatocellular Carcinoma.

OAB-019: CRISPR screens with single-cell transcriptome readout reveal potential mechanisms of response to natural killer cell treatment in multiple myeloma

<h3>Background</h3> Natural killer (NK) cell-based therapy is a promising approach to improve treatment responses in multiple myeloma (MM), which remains incurable. However, genetic determinants and mechanisms of sensitivity or resistance to NK cells in MM are incompletely understood. Here, we investigated the transcriptional impact of genes identified as regulators of sensitivity to NK cells in genome-wide CRISPR screens and integrated the findings with patient-derived data. <h3>Methods</h3> To systematically dissect the mechanisms by which select genes identified in our previous genome-wide studies influence MM cell response to NK cells, we performed pooled CRISPR screens with a single-cell (sc) transcriptome readout using the CROP-seq platform. Based on genome-wide screen ranking and biological relevance, 31 genes were selected. Pools of MM1.S and LP1 expressing sgRNAs targeting select hits were co-cultured with NK cells for 24 h or left untreated, followed by scRNA-seq and sgRNA detection and assignment to cells, followed by differential gene-expression analysis and patient data correlation. <h3>Results</h3> The analysis of the transcriptomic responses in MM cells upon NK cell exposure revealed that engagement by NK cells resulted in activation of IFNy-JAK-STAT signaling and upregulation of HLA class I genes, including the inhibitory NKG2A ligand HLA-E, as well as HLA class II. We then evaluated the impact of each sgRNA perturbation on gene expression in each condition, compared to the untreated control. Silencing of IFNy signaling mediators (JAK1, JAK2, STAT1, IFNGR2) prevented the induction of the IFNy response upon NK cell exposure, providing a mechanism for the observed sensitization to NK cells via reduced expression of HLA I genes. Disruption of TRAF2, NFKBIA, or NFKBIB, which also sensitized MM1.S to NK cells, induced NF-kB signaling. The induced NF-kB signature included increased expression of the death receptor FAS, pivotal for susceptibility to NK cells in the MM1.S CRISPR screen. We then correlated the findings with patient data from the CoMMpass dataset. Mutations in the NF-kB negative regulators TRAF2 and NFKBIA were associated with increased expression of NF-kB target genes also identified experimentally by CROP-seq. Also, NFKBIA mutations were linked to reduced HLA-E expression in both MM patients and CROP-seq, suggesting a potential explanation for the NK-sensitizing effect of NFKBIA disruption. Interestingly, patients with NLRC5 mutations had lower HLA-E expression consistent with CROP-seq data, indicating that although rare, NLRC5 mutations may predispose MM cells to higher NK-sensitivity. <h3>Conclusions</h3> Our data shed light on mechanisms of response to NK cells in MM and identify potential therapeutic targets for combination treatment. Furthermore, patient data correlation highlights subgroups that might have an increased susceptibility to NK cell treatment, highlighting the potential of such studies in the identification of predictive biomarkers.

A Closer Look at the Role of Anti-CCP Antibodies in the Pathogenesis of Rheumatoid Arthritis-Associated Interstitial Lung Disease and Bronchiectasis.

Rheumatoid arthritis (RA) is an articular disease with extra-articular manifestations. Pulmonary manifestations are not uncommon and can involve all compartments of the lungs with airway disease in the form of bronchiectasis or bronchiolitis, interstitial lung disease (ILD), pleural effusions and parenchymal lung nodules. The pulmonary features may present synchronously or after the articular disease, but, importantly, it may be the first presentation in 10% of patients in the absence of articular symptoms. Here we discuss the pathogenesis of RA lung involvement, particularly interstitial lung disease and bronchiectasis, focusing on the role anti-CCP antibodies (ACPAs). We highlight the complex interplay among genetic, environmental and immune factors. Furthermore, we explore the relationship of citrullination and smoking as well as the concept of interstitial pneumonia with autoimmune features (IPAF), where patients do not have evidence of another known cause of interstitial pneumonia and have incomplete features of connective tissue disease (CTD). We surmise that the frequency and titers of rheumatoid factor (RF) and ACPAs are increased in bronchiectasis and RA-bronchiectasis compared to RA patients without lung disease. ACPA is associated with more severe disease in both RA-ILD and RA-bronchiectasis even in the absence of articular symptoms. There is no clear prediction of development of articular RA with high ACPA levels in the context of positive ACPA and ILD; however, in RA-bronchiectasis, patients with positive antibodies can develop RA within a year after diagnosis of bronchiectasis. Though the primary focus of this narrative is to highlight the role of ACPA in pathogenesis and clinical practice, we also discuss the current treatment options and trials in RA-ILD and RA-bronchiectasis. Currently, there are no clear treatment guidelines. The treatments are now focusing on using a combination of immunosuppression and antifibrotic agents. Combination treatment targets both the fibrotic and inflammatory components of the disease process. Further studies are needed to identify the use of ACPA as a biomarker to tailor the treatment in RA-ILD and RA-bronchiectasis.

Identification of a novel therapeutic candidate, NRK, in primary cancer-associated fibroblasts of lung adenocarcinoma microenvironment.

Lung adenocarcinoma (LUAD) accounts for approximately half of patients in lung cancer. Cancer-associated fibroblasts (CAFs) are the major component in the tumor microenvironment (TME). Targeting CAFs is a promising therapeutic strategy for cancer treatment. However, therapeutic targets of CAFs in LUAD remains largely unclear. Seven CAFs and nine normal fibroblasts (NFs) were isolated from tumor and paratumor tissues of LUAD patients undergoing surgery, respectively. RNA-seq and bioinformatics analysis were performed to identify the differentially expressed genes (DEGs) and their functions in CAFs compared with NFs. DEGs of ten overlaying were obtained from RNA-seq, our previously reported lncRNA microarray and public datasets (E-MTAB-6149, E-MTAB-6653) and validated by RT-qPCR. Nik-related kinase (NRK) was further validated by RT-qPCR, immunofluorescence (IF), Western Blot (WB) in vitro, and in Cancer Cell Line Encyclopedia (CCLE) database. Survival analysis was performed on Kaplan-Meier plotter. A total of 1799 DEGs were identified, including 650 upregulated DEGs and 1149 downregulated DEGs. The upregulated and downregulated DEGs were mostly enriched in extracellular matrix (ECM) functions and in glycolysis/gluconeogenesis pathways. Interestingly, NRK was the most significantly upregulated overlaying DEGs which was rarely associated with CAFs before. NRK was predominantly expressed in CAFs, but weakly expressed in NFs, normal lung bronchial epithelial cell line BEAS-2B, LUAD cell lines A549 and H1299, as well as in the majority of 191 lung cancer cell lines including LUAD. Moreover, elevated NRK predicted poor survival in LUAD patients. Here, we first report that NRK is significantly elevated in LUAD-associated CAFs and may function as a promising therapeutic target for cancer combination treatment. Besides, modulation of ECM and glycolysis/gluconeogenesis pathways may be an efficient approach to alter CAFs functionality in LUAD.

Tumor-promoting macrophages prevail in malignant ascites of advanced gastric cancer

Gastric cancer (GC) patients develop malignant ascites as the disease progresses owing to peritoneal metastasis. GC patients with malignant ascites have a rapidly deteriorating clinical course with short survival following the onset of malignant ascites. Better optimized treatment strategies for this subset of patients are needed. To define the cellular characteristics of malignant ascites of GC, we used single-cell RNA sequencing to characterize tumor cells and tumor-associated macrophages (TAMs) from four samples of malignant ascites and one sample of cerebrospinal fluid. Reference transcriptomes for M1 and M2 macrophages were generated by in vitro differentiation of healthy blood-derived monocytes and applied to assess the inflammatory properties of TAMs. We analyzed 180 cells, including tumor cells, macrophages, and mesothelial cells. Dynamic exchange of tumor-promoting signals, including the CCL3–CCR1 or IL1B–IL1R2 interactions, suggests macrophage recruitment and anti-inflammatory tuning by tumor cells. By comparing these data with reference transcriptomes for M1-type and M2-type macrophages, we found noninflammatory characteristics in macrophages recovered from the malignant ascites of GC. Using public datasets, we demonstrated that the single-cell transcriptome-driven M2-specific signature was associated with poor prognosis in GC. Our data indicate that the anti-inflammatory characteristics of TAMs are controlled by tumor cells and present implications for treatment strategies for GC patients in which combination treatment targeting cancer cells and macrophages may have a reciprocal synergistic effect.

MiR-146a-5p impairs melanoma resistance to kinase inhibitors by targeting COX2 and regulating NFkB-mediated inflammatory mediators

BackgroundTargeted therapy with BRAF and MEK inhibitors has improved the survival of patients with BRAF-mutated metastatic melanoma, but most patients relapse upon the onset of drug resistance induced by mechanisms including genetic and epigenetic events. Among the epigenetic alterations, microRNA perturbation is associated with the development of kinase inhibitor resistance. Here, we identified and studied the role of miR-146a-5p dysregulation in melanoma drug resistance.MethodsThe miR-146a-5p-regulated NFkB signaling network was identified in drug-resistant cell lines and melanoma tumor samples by expression profiling and knock-in and knock-out studies. A bioinformatic data analysis identified COX2 as a central gene regulated by miR-146a-5p and NFkB. The effects of miR-146a-5p/COX2 manipulation were studied in vitro in cell lines and with 3D cultures of treatment-resistant tumor explants from patients progressing during therapy.ResultsmiR-146a-5p expression was inversely correlated with drug sensitivity and COX2 expression and was reduced in BRAF and MEK inhibitor-resistant melanoma cells and tissues. Forced miR-146a-5p expression reduced COX2 activity and significantly increased drug sensitivity by hampering prosurvival NFkB signaling, leading to reduced proliferation and enhanced apoptosis. Similar effects were obtained by inhibiting COX2 by celecoxib, a clinically approved COX2 inhibitor.ConclusionsDeregulation of the miR-146a-5p/COX2 axis occurs in the development of melanoma resistance to targeted drugs in melanoma patients. This finding reveals novel targets for more effective combination treatment.4ZiY3nqT8xQ_yvPXVejWjwVideo Graphical

Renal Cell and Urothelial Carcinoma: Biomarkers for New Treatments.

Therapies for genitourinary malignancies have evolved considerably in the past five years. Combination treatment targeting biologically relevant immune and angiogenic pathways is improving patient survival in metastatic renal cell carcinoma (RCC), whereas immune checkpoint blockade (ICB), novel targeted therapy, and antibody drug conjugates have changed the landscape of urothelial cancer (UC) treatment. A daily challenge for clinicians is identifying patients who derive a preferential benefit from the available therapeutic options. The completion of large-scale genomics projects has yielded comprehensive descriptions of the molecular heterogeneity present in RCC and UC, although clinical applications of these data continue to evolve. Major molecular subtypes of RCC align well with histology subtype, and although some molecular characteristics appear to carry prognostic information, biomarkers predicting benefit from tyrosine kinase inhibitor (TKI) or immunotherapy are generally lacking. Unexpectedly, similar work has demonstrated that UC can be grouped into "molecular subtypes" that share properties with those found in breast cancer and other solid tumors. Furthermore, this molecular subtype classification is prognostic and potentially predictive of differential benefit from conventional and targeted therapies. This article provides an update on the current state of molecular biomarker development and potential clinical utility in RCC and UC.

Phase Ib/II umbrella trial to evaluate the safety and efficacy of multiple 2L cancer immunotherapy (CIT) combinations in advanced/metastatic urothelial carcinoma (mUC): MORPHEUS-mUC.

TPS591 Background: Although CIT has efficacy against multiple cancers, only subsets of patients (pts) have durable responses with monotherapy. CIT combination treatment (tx) targets multiple immune-evasion mechanisms and may thus be more likely to improve outcomes. However, a large number of potential combinations need to be tested to identify effective combinations. The MORPHEUS platform comprises multiple global, open-label, randomized Phase Ib/II trials to identify early efficacy signals and safety of tx combinations in pts with different cancers. MORPHEUS-mUC is a randomized study of 6 CIT combinations compared with a single CIT control in pts with locally advanced UC/mUC who progressed during or after a platinum-containing regimen. The PD-L1 inhibitor atezolizumab will be combined with agents targeting different mechanisms (via nectin-4, PARP, CD47, CD38, DPP-4 and IL-6R) to simultaneously enhance immune cell priming and activation, tumor infiltration and/or recognition of tumor cells. To accelerate the clinical development of novel combinations, MORPHEUS has the flexibility, via amendment, to allow for the modification of eligibility criteria or the opening of new tx arms to replace arms that demonstrate no signs of clinical activity/unacceptable toxicity. Methods: MORPHEUS-mUC (NCT03869190) has 2 stages. In Stage 1, approximately 130-305 pts with histologically documented, locally advanced inoperable UC/mUC will be randomized to atezolizumab monotherapy (control) or experimental arms: atezolizumab + either enfortumab vedotin, niraparib, magrolimab (Hu5F9-G4), isatuximab, linagliptin or tocilizumab. Safety will be monitored for potential overlapping toxicities. Stage 2 arms will be atezolizumab + either enfortumab vedotin or linagliptin arms, unless these combinations show no activity in Stage 1. Eligible pts who experience unacceptable toxicity or loss of clinical benefit in Stage 1 may enroll in Stage 2 within 3 months. The primary study endpoint is investigator-assessed ORR per RECIST 1.1; secondary endpoints include PFS, OS, DOR, DCR and safety. Biomarkers will also be explored. Clinical trial information: NCT03869190.