Activation Of Oncogenic Pathways Research Articles

Abstract P5-08-06: MDX-124, a novel annexin-A1 antibody, shows anti-tumor efficacy in several preclinical models of triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) accounts for 10-15% of all breast cancer cases and is a particularly aggressive form of the disease, with poorer prognosis compared to other breast cancer subtypes. Advances in TNBC treatment have been made with the introduction of checkpoint inhibitors, PARP inhibitors and antibody drug conjugates. However, these therapies still have several limitations including low patient response rates, treatment resistance or relapse, and considerable side effects. Therefore, a significant unmet clinical need remains to develop novel targeted agents to treat TNBC. Annexin-A1 (ANXA1) is a phospholipid binding protein secreted in response to several physiological stimuli where it activates formyl peptide receptors (FPR1/2) triggering multiple oncogenic cell signaling pathways. Overexpression of ANXA1 by TNBC cells has been shown to influence several cancer-related processes including cell growth, cell cycle progression, angiogenesis, migration and invasion. In addition, ANXA1 has been shown to have immunomodulatory effects on T-cells, macrophages and dendritic cells. High ANXA1 expression in TNBC patients correlates with both poor overall survival and progression-free survival, indicating ANXA1 is a therapeutic target in TNBC. MDX-124 is a novel humanized antibody targeting ANXA1, and we have previously demonstrated its significant anti-proliferative activity. Here we present additional data showing the efficacy of MDX-124 in preclinical models of TNBC. Methods: The impact of MDX-124 on cell cycle progression was evaluated by measuring changes in DNA content using flow cytometry and analyzed with FlowJo™ software.The modulation of key oncogenic cell signalling pathways by MDX-124 was assessed using human phospho-kinase and XL Oncology Proteome Profiler™ antibody arrays. The effect of MDX-124 in combination therapy with chemotherapy was studied in-vitro using MTT cell proliferation assays, whilst synergy with anti-PD-1 therapy was evaluated using the EMT6 syngeneic mouse model of TNBC. Results: MDX-124 treatment dramatically decreased the proportion of HCC1806 TNBC cells in S-phase by 29.1% with a concomitant increase in G1 of 33.5% versus untreated cells. This occurred in a dose-dependent manner and is consistent with an MDX-124 mediated increase in cell cycle arrest.Treatment of HCC1806 TNBC cells with MDX-124 altered the expression of key oncogenic proteins and the phosphorylation of several kinases that regulate cell signaling pathways involved in proliferation, survival and migration. Notably, MDX-124 substantially reduced ERK and AKT phosphorylation by 84% and 72% respectively versus untreated control cells. In the HCC1806 TNBC cell line, proliferation was significantly reduced after 72 h treatment with MDX-124 and chemotherapy versus control.In the EMT6 syngeneic mouse model of TNBC, the murine analogue of MDX-124 (MDX-001) potentiated mean tumor growth inhibition of single agent anti-PD-1 treatment by 15%. Additionally, 30% of treated mice showed tumor regression in the MDX-001 combination therapy group versus 10% in the single agent anti-PD-1 group. This suggest that anti-ANXA1 antibody therapy acts synergistically with anti-PD-1 immunotherapy.Conclusion: MDX-124 binds to secreted and extracellular ANXA1 disrupting interactions with FPR1/2. This results in altered expression levels of several key cancer-related proteins preventing the activation of oncogenic signaling pathways that promote cancer progression. MDX-124 has demonstrated anti-cancer activity in several TNBC cell line and mouse models, as both a single agent and in combination with other drugs, including anti-PD-1 immunotherapy. Medannex plan to initiate a First-In-Human study in Q4 2021 to evaluate MDX-124 in solid malignancies, including TNBC. Citation Format: Fiona C Dempsey, Hussein Al-Ali, Scott J Crichton, Charlene Fabian, Chris Pepper, Christopher N Parris. MDX-124, a novel annexin-A1 antibody, shows anti-tumor efficacy in several preclinical models of triple-negative 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 P5-08-06.

Decoding Insulin-Like Growth Factor Signaling Pathway From a Non-coding RNAs Perspective: A Step Towards Precision Oncology in Breast Cancer.

Breast cancer (BC) is a highly complex and heterogenous disease. Several oncogenic signaling pathways drive BC oncogenic activity, thus hindering scientists to unravel the exact molecular pathogenesis of such multifaceted disease. This highlights the urgent need to find a key regulator that tunes up such intertwined oncogenic drivers to trim the malignant transformation process within the breast tissue. The Insulin-like growth factor (IGF) signaling pathway is a tenacious axis that is heavily intertwined with BC where it modulates the amplitude and activity of vital downstream oncogenic signaling pathways. Yet, the complexity of the pathway and the interactions driven by its different members seem to aggravate its oncogenicity and hinder its target-ability. In this review, the authors shed the light on the stubbornness of the IGF signaling pathway and its potential regulation by non-coding RNAs in different BC subtypes. Nonetheless, this review also spots light on the possible transport systems available for efficient delivery of non-coding RNAs to their respective targets to reach a personalized treatment code for BC patients.

Generation of mice for evaluating endogenous p16Ink4a protein expression

The cyclin-dependent kinase inhibitor p16Ink4a plays a central role in cellular senescence in vitro. Although previous studies suggested cellular senescence is integrated in the systemic mechanisms of organismal aging, the localization and the dynamics of p16Ink4a in tissues remain poorly understood, which hinders uncovering the role of p16Ink4a under the in vivo context. One of the reasons is due to the lack of reliable reagents; as we also demonstrate here that commonly used antibodies raised against human p16INK4A barely recognize its murine ortholog. Here we generated a mouse model, in which the endogenous p16Ink4a is HA-tagged at its N-terminus, to explore the protein expression of p16Ink4a at the organismal level. p16Ink4a was induced at the protein level along the course of senescence in primary embryonic fibroblasts derived from the mice, consistently to its transcriptional level. Remarkably, however, p16Ink4a was not detected in the tissues of the mice exposed to pro-senescence conditions including genotoxic stress and activation of oncogenic signaling pathways, indicating that there is only subtle p16Ink4a proteins induced. These results in our mouse model highlight the need for caution in evaluating p16Ink4a protein expression in vivo.

Splicing is an alternate oncogenic pathway activation mechanism in glioma

High-grade diffuse glioma (HGG) is the leading cause of brain tumour death. While the genetic drivers of HGG have been well described, targeting these has thus far had little impact on survival suggesting other mechanisms are at play. Here we interrogate the alternative splicing landscape of pediatric and adult HGG through multi-omic analyses, uncovering an increased splicing burden compared with normal brain. The rate of recurrent alternative splicing in cancer drivers exceeds their mutation rate, a pattern that is recapitulated in pan-cancer analyses, and is associated with worse prognosis in HGG. We investigate potential oncogenicity by interrogating cancer pathways affected by alternative splicing in HGG; spliced cancer drivers include members of the RAS/MAPK pathway. RAS suppressor neurofibromin 1 is differentially spliced to a less active isoform in >80% of HGG downstream from REST upregulation, activating the RAS/MAPK pathway and reducing glioblastoma patient survival. Overall, our results identify non-mutagenic mechanisms by which cancers activate oncogenic pathways which need to accounted for in personalized medicine approaches.

Comprehensive Molecular Characterization Reveals Genomic and Transcriptomic Subtypes of Metastatic Urothelial Carcinoma

Recent molecular characterization of primary urothelial carcinoma (UC) may guide future clinical decision-making. For metastatic UC (mUC), a comprehensive molecular characterization is still lacking. We analyzed whole-genome DNA and RNA sequencing data for fresh-frozen metastatic tumor biopsies from 116 mUC patients who were scheduled for palliative systemic treatment within the context of a clinical trial (NCT01855477 and NCT02925234). Hierarchical clustering for mutational signatures revealed two major genomic subtypes: GenS1 (67%), which was APOBEC-driven; and GenS2 (24%), which had a high fraction of de novo mutational signatures related to reactive oxygen species and is putatively clock-like. Significantly mutated genes (SMGs) did not differ between the genomic subtypes. Transcriptomic analysis revealed five mUC subtypes: luminal-a and luminal-b (40%), stroma-rich (24%), basal/squamous (23%), and a nonspecified subtype (12%). These subtypes differed regarding expression of key genes, SMGs, oncogenic pathway activity, and immune cell infiltration. We integrated the genomic and transcriptomic data to propose potential therapeutic options by transcriptomic subtype and for individual patients. This in-depth analysis of a large cohort of patients with mUC may serve as a reference for subtype-oriented and patient-specific research on the etiology of mUC and for novel drug development. Patient summaryWe carried out an in-depth analysis of the molecular and genetic features of metastatic cancer involving the cells that line the urinary tract. We showed that this is a heterogeneous disease with different molecular subtypes and we identified possible targets for therapy for each subtype.

Novel inhibitor of hematopoietic cell kinase as a potential therapeutic agent for acute myeloid leukemia.

Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are characterized by risk of relapses, poor survival, unwanted side effects and high toxicity with the current therapies. In light of these facts, there are efforts to develop new drugs specific for deregulated molecules that participate in leukemia pathogenesis. Hematopoietic cell kinase (HCK), an Src kinase family member, is overexpressed on hematopoietic stem cells of MDS and de novo AML patients and involved in the oncogenic process. Thus, we investigated in vitro, ex vivo and in vivo effects of a novel chemical compound targeting HCK inhibition (iHCK-37), in combination with the most used drugs for the treatment of MDS and de novo AML, 5-Azacytidine and Cytarabine. Herein, the combination treatment with iHCK-37 and 5-Azacytidine or Cytarabine demonstrated additive effects against leukemia cells, compared to either drug alone. iHCK-37 plus 5-Azacytidine or Cytarabine treatment was able to reduce the activation of oncogenic pathways, MAPK/ERK and PI3K/AKT, leading to reduction of ERK and AKT phosphorylation, and increased BAX and decreased BCL-XL protein expression. Moreover, treatment with iHCK-37 reduced MDS and AML CD34-positive cell numbers inside a 3D-structure but did not affect normal CD34-positive cell numbers. In vivo analysis showed that leukemic mice treated with iHCK-37 had reduced ERK and AKT proteins phosphorylation levels and leukocyte numbers. In conclusion, the iHCK-37 inhibitor has anti-neoplastic activity in leukemia cells without altering apoptosis and survival rate of normal cells, suggesting on-target malignant cell killing activity as a single agent or in combination with 5-Azacytidine or Cytarabine.

Abstract P201: Zenocutuzumab is an effective HER2/HER3 Biclonics® antibody in cancers with NRG1 fusions

Abstract Neuregulin 1 (NRG1) fusion proteins are oncogenic drivers in multiple cancer types, including non-small cell lung cancer, pancreas adenocarcinoma, and other solid tumors. NRG1 fusion proteins bind to HER3 and signal through HER2/HER3 heterodimers, leading to oncogenic transformation. Zenocutuzumab is an HER2/HER3 humanized IgG1 bispecific antibody that specifically and potently blocks NRG1 fusion-driven signaling of the heterodimeric complex. Zenocutuzumab binds via its anti-HER2 monovalent Fab arm to domain I of HER2. Docking of zenocutuzumab in this position increases the effective affinity of the anti-HER3 Fab arm, which binds to domain 3, a critical epitope that blocks binding of the HER3 ligand NRG1. Consequently, zenocutuzumab displaces NRG1 binding to HER3, even at high ligand concentrations, keeping HER3 in an inactive state and blocking HER2/HER3 heterodimerization and NRG1-mediated signaling. The clinical efficacy and safety of zenocutuzumab were recently demonstrated in patients with NRG1 fusion-driven cancers who were enrolled in the ongoing global multicenter eNRGy study and a global early access program NCT02912949 (J Clin Oncol 39;15_suppl; abstract 3003). Treatment with zenocutuzumab led to rapid and major radiologic tumor regression and biomarker responses in heavily pretreated patients with multiple NRG1 fusion-positive cancers with several different N-terminal fusion partners. Zenocutuzumab has been enhanced for antibody-dependent cell-mediated cytotoxicity (ADCC) to recruit and bolster immune effector cells to aid in tumor elimination. Here we explored the different Fc-mediated immune effector functions of zenocutuzumab. In several cancer cell lines, zenocutuzumab induced Fc-mediated dose-dependent ADCC and antibody-dependent cellular phagocytosis. Insertion of ATP1B1-NRG1 and SLC3A2-NRG1 into immortalized pancreatic ductal epithelial cells (H6c7) led to activation of the HER3 oncogenic signaling pathways including AKT. The H6c7-SLC3A2-NRG1 cells formed tumors when implanted in the subcutaneous flank of immune compromised mice (after about 10 weeks). Furthermore, a dependency of the transformed cell on this oncogene for growth and survival was also observed. Zenocutuzumab effectively inhibited proliferation of these NRG1 fusion-expressing, transformed cells. In addition, proteomic analysis showed that zenocutuzumab inhibited the HER3 and AKT-related downstream signaling pathways. In conclusion, these data provide strong mechanistic evidence supporting the clinical efficacy of zenocutuzumab in patients with NRG1 fusion-driven cancers. Citation Format: Jan Gerlach, Igor Odintsov, Ron Schackmann, Marc Ladanyi, Jeroen Lammerts van Bueren, Romel Somwar, Cecile Geuijen. Zenocutuzumab is an effective HER2/HER3 Biclonics® antibody in cancers with NRG1 fusions [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 P201.

Abstract P114: Engineered hydrogel elucidates contributions of matrix mechanics to esophageal adenocarcinoma and identify matrix-activated therapeutic targets

Abstract Changes in the tumor microenvironment arbitrated by a stiffened ECM are associated with tumor aggression and enable increased propensity towards metastasis. For instance, in vitro (2D) studies have implicated ECM properties in EAC progression, suggesting their exploitation for esophageal therapy following surgical resection. However, these studies are limited by the lack of 3D intercellular interactions, underscoring the need for physiologically relevant 3D culture models (e.g., organoids) that better recapitulate human cancer and its microenvironment to elucidate underlying mechanisms. Engineered hydrogels are an evolving and important component of 3D organoid culture systems, especially to introduce tunable physicochemical matrix signals that have been investigated in tumor progression and metastasis. Furthermore, patient-derived tumor organoids have become an attractive pre-clinical in vitro model to study cancer biology and evaluate response to therapeutics. Therefore, we have engineered a visible light-mediated hydrogel platform that supports the development of patient-derived Barrett’s esophagus (BE) organoids, a precursor to esophageal adenocarcinoma (EAC), as well as EAC organoids. This synthetic biomaterial platform allows control over hydrogel stiffness to better recapitulate the mechanically dynamic esophageal cancer microenvironment, and may help identify therapeutic targets in EAC organoids. Our preliminary data have demonstrated that BE and EAC organoid density, size and proliferation can be controlled by synthetic ECM biomechanical properties, demonstrating the significance of understanding the independent contributions of ECM properties to EAC development, which is inherently limited in biologically-derived materials (e.g. MatrigelTM). Furthermore, our data show that increased hydrogel stiffness promotes increased expression of mutant TP53, and increased SOX9 expression as a result of YAP1 activation, suggesting that matrix mechanics have a significant role in conferring stem-like properties to EAC organoids via activation of canonical oncogenic signaling pathways. Ongoing studies involve identifying matrix-activated therapeutic targets via small-molecule inhibition of TP53 and YAP1, as a function of matrix stiffening, and identifying differentially expressed genes using high-throughput transcriptome sequencing between stiffened hydrogel-encapsulated EAC organoid conditions. Our work is significant because it establishes a biomaterial platform that overcomes the limitations of current 3D organoid culture methods to elucidate the role of the tumor microenvironment in EAC tumorigenesis and to identify disease-relevant therapeutic targets. Successful completion of this work will also provide an opportunity to further establish the engineered biomaterial as a platform to potentially elucidate the mechanisms of, and therapy targets for, other human adenocarcinomas in the context of changes in the mechanics of tumor microenvironment. Citation Format: Ricardo Cruz-Acuña, Claudia Loebel, Jason A. Burdick, Anil K. Rustgi. Engineered hydrogel elucidates contributions of matrix mechanics to esophageal adenocarcinoma and identify matrix-activated therapeutic targets [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 P114.

PRRC2A Promotes Hepatocellular Carcinoma Progression and Associates with Immune Infiltration

PurposeHepatocellular carcinoma (HCC) has high morbidity and poor prognosis due to the propensity of recurrence and metastasis. Emerging studies have confirmed that proline-rich coiled-coil2A (PRRC2A) plays a crucial role in tumorigenesis and immunoregulation. However, its expression status and biological functions in HCC remain poorly documented.MethodsThe presence and prognostic value of PRRC2A were determined by a tissue microarray (TMA) cohort and multiple databases, mainly from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), Clinical Proteomic Tumor Analysis Consortium (CPTAC). Functional enrichment analysis was applied to identify the mechanisms of PRRC2A in HCC. The biological function of PRRC2A in HCC progression in vitro was determined by CCK-8, colony formation, EdU, transwell migration and invasion assays. Moreover, the Estimation of STromal and Immune cells in Malignant Tumor tissues using Expression data (ESTIMATE), single-sample gene set enrichment analysis (ssGSEA), tumor immune dysfunction and exclusion (TIDE) algorithms, immunophenoscore (IPS) and public available immunotherapy cohorts were performed to classify their associations with tumor-infiltrating immune cells and immunotherapy.ResultsPRRC2A was upregulated in HCC at both mRNA and protein levels. High PRRC2A expression was correlated with poor prognosis and could be an independent risk factor. Functional enrichment analysis demonstrated that elevated PRRC2A was significantly correlated with the activation of various oncogenic pathways. Additionally, in vitro experiments confirmed that silencing PRRC2A could suppress the proliferation and metastasis capacities of HCC cells. More importantly, PRRC2A was negatively associated with many anti-tumor immune cells, but positively related to the expression of markers of exhaustive T cells. And HCC patients with high PRRC2A were more likely to be nonresponsive to immunotherapy.ConclusionThis study explored the predictive value and biological roles of PRRC2A in HCC progression and indicated that it might be a potential biomarker for HCC patients and a predictor for immunotherapy.

Leveraging Pathway-Interference to Overcome Drug-Resistance in Acute Lymphoblastic Leukemia

Background: The concept of multi-step carcinogenesis (Fearon and Vogelstein 1990) suggests that acquisition of mutations in addition to an existing set of mutations invariably accelerates tumor-progression. In colorectal cancer and many other cancer types, activation of multiple distinct oncogenic pathways is required for the development of invasive cancer. Here, we examined this paradigm for genetic lesions in B-ALL and 13 other cancer types.Bioinformatic approaches: To broadly study how oncogenic drivers across multiple signaling pathways interact, we developed a bioinformatic platform to map interactions between genetic lesions that cause oncogenic activation of eight oncogenic pathways, including PI3K, STAT5, NF-κB, Hippo, Notch, WNT, RAS-ERK and TGFβ-Smad pathways. Plotting of interaction scores between 56 pathway pairs in a matrix for 14 cancer types revealed that 12 of the 14 cancer types showed a pattern of globally synergistic pathway interactions, consistent with the Fearon and Vogelstein model of cooperation of multiple pathways to drive malignant transformation. Strikingly, B-ALL and gliomas showed the opposite behavior with largely antagonistic pathway interactions.Results. Unlike the vast majority of cancer types, B-ALL and gliomas are driven by one principal oncogenic pathway at a time. While the reasons for negative pathway interactions in glioma are unknown, we focused on functional analyses on pathway interference patterns in 1,148 cases of B-ALL. Genetic lesions leading to STAT5- or ERK-pathway activation are frequently found in B-ALL. Interestingly, activating lesions of both pathways co-occurred in only 3% of the cases studied, suggesting that co-activation of STAT5- and ERK-occurs much less frequently than expected by chance (odds ratio 0.13, P=2e-16, Figure, left). Unbiased interaction mapping analyses of mutational co-occurrence indicated strong negative selection for dual activation of both STAT5- and ERK-pathways (Figure, middle). Importantly this inter-pathway aversion is much stronger than intra-pathway effects, reflecting incompatibility rather than redundancy. Even in rare cases of co-occurrence in the same sample, single-cell mutation and phosphoprotein analyses revealed that STAT5- and ERK-activating mutations were mutually exclusive and reflected two competing clones.STAT5- and ERK-pathways engage conflicting transcriptional and biochemical programs, resulting in “friction”, when both pathways are concurrently activated. In agreement with pathway interference, we demonstrated that Cre-mediated deletion of divergent pathway components - Erk2 fl/fl in a STAT5-driven model of B-ALL and Stat5 fl/fl in an ERK-driven B-ALL model - dramatically accelerated initiation of fatal leukemia in vivo. While Cre-mediated deletion of divergent pathway components precipitated leukemia-initiation, these findings suggest that reactivation of divergent signaling pathways represents a powerful barrier against malignant transformation.Interestingly, our preclinical studies suggested that pharmacological reactivation of divergent (suppressed) pathways can be leveraged for therapeutic benefit: The DUSP6 small molecule inhibitor BCI-215 functions as powerful activator of ERK and suppresses STAT5-phosphorylation, i.e. the principal pathway in STAT5-driven B-ALL (Figure, right). Likewise, DPH, a small molecule STAT5-agonist interferes with ERK-phosphorylation, the principal oncogenic driver in RAS-pathway B-ALL (Figure, right). Both BCI-215 and DPH significantly prolonged overall survival of NSG mice transplanted with refractory STAT5- and ERK-driven B-ALL PDX, respectively.Conclusions: We propose that a diverse spectrum of signaling input reflects interactions of normal cells with their environment, while convergence on one centralized pathway is a hallmark of cancer. Tracking early stages of leukemia-initiation, we identified convergence on one principal oncogenic driver and inactivation of diverging pathways as an early critical step. Pharmacological reactivation of divergent signaling pathways to subvert transformation was achievable by STAT5- and ERK-agonists. Proof-of-concept studies in patient-derived B-ALL cells revealed that pharmacological reactivation of suppressed divergent circuits can be leveraged as a previously unrecognized strategy to overcome drug-resistance. [Display omitted] DisclosuresIzraeli: Roche: Consultancy, Speakers Bureau; Bayer: Speakers Bureau; sightDx: Consultancy. Weinstock: ASELL: Consultancy; SecuraBio: Consultancy; Bantam: Consultancy; AstraZeneca: Consultancy; Abcuro: Research Funding; Verastem: Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Travera: Other: Founder/Equity; Ajax: Other: Founder/Equity.

TP53 Alterations Enrich in Ezb/C3-like Subclassified Relapsed or Refractory DLBCL and Are Susceptible to BRD4 Protac Inhibition

Introduction: New Diffuse Large B-cell (DLBCL) treatments remain a clinical need despite the success of rituximab combined with CHOP chemotherapy (RCHOP), which results in durable responses in 60-70% of patients. Those refractory to or who relapse following first-line therapy (rrDLBCL) have a very poor outcome, with only 20% surviving beyond 5 years, although CAR-T cell therapy is making a significant impact. Genomic insights can inform mechanism and treatment studies within this population, specifically the precision application of CAR-T. Rushton et. al. recently analyzed a large rrDLBCL patient cohort, noting significant rises in specific genes during the transition to advanced disease (MS4A1, TP53, NFKBIE, FOXO1, and CREBBP), building on the innovations of pre-treatment classifications. Addressing the heterogeneous activation of oncogenic pathways remains a challenge. Inhibitors targeting genes responsible for epigenetic regulation of rrDLBCL represent an attractive option, namely the transcriptional regulator BRD4, with PROTAC inhibition resulting in downregulation of several key DLBCL oncogenes. Herein, we report the first genomic subclassifications of rrDLBCL, the enrichment of TP53 alterations in the EZB-like subclassification, and data supporting the application of BRD4-targeting PROTAC therapies.Methods: Non-negative matrix factorization (NMF) was applied to a publicly available cohort of 127 RCHOP-treated rrDLBCL patients with matching sequencing data. Marker selection was applied to the 2 emergent groups to detect differential presence of DNA alterations. A Pearson similarity matrix was applied to the rrDLBCL cohort and 3 pretreatment cohorts to detect patterns of DNA alteration association and exclusion. Next, CRISPR-KO gene scores were integrated against gene expression fold change values before and after BRD4 PROTAC treatment. Lasty, 4 DLBCL cell lines were assayed against the BRD4 PROTAC ARV-825, Venetoclax, and inhibitors of epigenetic regulation.Results: NMF produced 2 clusters based on DNA alterations from the rrDLBCL cohort: RR1 and RR2. Significantly associated genes (FDR ≤ 0.05) included MYD88, PIM1, IGLL5, HIST1HC, SOCS1, HIST1H1E, and CD79B in RR1 , and CREBBP, EZH2, STAT6, BCL2, TP53, TNFRSF14, CARD11, and double hit translocations in RR2. Notably, TP53 alterations significantly enriched in the EZB subclassification during rrDLBCL transition in comparison to pre-treatment distribution (P = 0.0016). Significant associations between TP53 and EZB genes were observed alongside significant exclusion from MCD and BN2-associated genes. Integrating significant gene expression changes following BRD4-PROTAC treatment and CRISPR-KO scores revealed the loss of several key oncogenes in cell lines, including BCL2, BCL6, CD79B, POU2F2, and MDM4. Markedly, reductions in 9 established GCB and 4 ABC gene signatures were observed alongside the reduction of the Reactome signature of TP53 Regulators of Expression and Degradation (FDR = 0.0377). In vitro inhibition assays measuring ARV-825 (BRD4 PROTAC) response revealed significantly lower rates of cell viability compared to Venetoclax across 4 cell lines.Conclusions: Our results indicate that distinct genomic groups exist within rrDLBCL, that TP53 alterations strongly associate with the EZB-enriched group, and that if TP53 alterations continue to predict poor CAR-T response, these tumors may be susceptible to BRD4 inhibition. Our observations indicate a significant climb in TP53 association with EZB tumors and their matched alterations after rrDLBCL transition in comparison to the pre-treatment landscape. Enrichment may indicate that EZB-like tumors lack some of the tools necessary to make the transition to advanced disease but provide an ideal environment for the acquisition of TP53 alterations. In contrast, the disassociation between TP53 alterations and MCD/BN2-like alterations may indicate that these tumors have more inherent capability to reject therapy. The emergence of BRD4-targeting PROTACs provides an additional clinical tool, one perhaps more suitable than Venetoclax against specific rrDLBCL cases. In closing, This study provides insights towards the rrDLBCL landscape and reinforces the necessity of evaluating tissue biopsies for TP53 alterations throughout treatment to guide precision application of therapy, noting susceptible pathways of these particular tumors. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Mechanisms of Resistance to CDK4/6 Blockade in Advanced Hormone Receptor-positive, HER2-negative Breast Cancer and Emerging Therapeutic Opportunities.

The cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) have become the standard of care, in combination with antiestrogen therapy, for patients with hormone receptor-positive (HR+)/HER2- advanced breast cancer. Various preclinical and translational research efforts have begun to shed light on the genomic and molecular landscape of resistance to these agents. Drivers of resistance to CDK4/6i therapy can be broadly subdivided into alterations impacting cell-cycle mediators and activation of oncogenic signal transduction pathways. The resistance drivers with the best translational evidence supporting their putative role have been identified via next-generation sequencing of resistant tumor biopsies in the clinic and validated in laboratory models of HR+ breast cancer. Despite the diverse landscape of resistance, several common, therapeutically actionable resistance nodes have been identified, including the mitotic spindle regulator Aurora Kinase A, as well as the AKT and MAPK signaling pathways. Based upon these insights, precision-guided therapeutic strategies are under active clinical development. This review will highlight the emerging evidence, in the clinic and in the laboratory, implicating this diverse spectrum of molecular resistance drivers.

Genetic and epigenetic insights into cutaneous T-cell lymphoma

AbstractPrimary cutaneous T-cell lymphomas (CTCLs) constitute a heterogeneous group of non-Hodgkin T-cell lymphomas that present in the skin. In recent years, significant progress has been made in the understanding of the pathogenesis of CTCLs. Progress in CTCL classifications combined with technical advances, in particular next-generation sequencing, enabled a more detailed analysis of the genetic and epigenetic landscape and transcriptional changes in clearly defined diagnostic entities. These studies not only demonstrated extensive heterogeneity between different CTCL subtypes but also identified recurrent alterations that are highly characteristic for diagnostic subgroups of CTCLs. The identified alterations, in particular, involve epigenetic remodeling, cell cycle regulation, and the constitutive activation of targetable oncogenic pathways. In this respect, aberrant JAK-STAT signaling is a recurrent theme; however, it is not universal for all CTCLs and has seemingly different underlaying causes in different entities. A number of the mutated genes identified are potentially actionable targets for the development of novel therapeutic strategies. Moreover, these studies have produced an enormous amount of information that will be critically important for the further development of improved diagnostic and prognostic biomarkers that can assist in the clinical management of patients with CTCL. In the present review, the main findings of these studies in relation to their functional impact on the malignant transformation process are discussed for different subtypes of CTCLs.

Identification of Novel Molecular Therapeutic Targets and Their Potential Prognostic Biomarkers Among Kinesin Superfamily of Proteins in Pancreatic Ductal Adenocarcinoma.

BackgroundKinesin superfamily of proteins (KIFs) has been broadly reported to play an indispensable role in the biological process. Recently, emerging evidence reveals its oncogenic role in various cancers. However, the prognostic, oncological, and immunological values of KIFs have not been comprehensively explored in pancreatic ductal adenocarcinoma (PDAC) patients. We aimed to illustrate the relationship between KIFs and pancreatic ductal adenocarcinoma by using bioinformatical analysis.MethodsWe use GEPIA, Oncomine datasets, cBioPortal, LOGpc, TIMER, and STRING bioinformatics tools and web servers to investigate the aberrant expression, prognostic values, and oncogenic role of KIFs. The two-gene prognostic model and the correlation between KIFs and KRAS and TP53 mutation were performed using an R-based computational framework.ResultsOur results demonstrated that KIFC1/2C/4A/11/14/15/18A/18B/20B/23 (we name it prognosis-related KIFs) were upregulated and associated with unfavorable clinical outcome in pancreatic cancer patients. KIF21B overexpression is associated with better clinical outcome. The KIFC1/2C/4A/11/14/15/18A/18B/20B/23 profiles were significantly increased compared to grade 1 and grade 2/3. Besides, KIFC1/2C/4A/11/14/15/18A/18B/20B/23 was significantly associated with the mutation status of KRAS and TP53.Notably, most prognosis-related KIFs have strong correlations with tumor growth and myeloid-derived suppressor cells infiltration (MDSCs). A prognostic signature based on KIF20B and KIF21B showed a reliable predictive performance. Receiver operating characteristic (ROC) curve was employed to assess the predictive power of two-gene signature. Consequently, the gene set enrichment analysis (GSEA) showed that KIF20B and KIF21B’s overexpression was associated with the immunological and oncogenic pathway activation in pancreatic cancer. Finally, real-time quantitative PCR (RT-qPCR) was utilized to investigate the expression pattern of KIF20B and KIF21B in pancreatic cancer cell lines and normal pancreatic cell.ConclusionsKnowledge of the expression level of the KIFs may provide novel therapeutic molecular targets and potential prognostic biomarkers to pancreatic cancer patients.

DNA damage responses that enhance resilience to replication stress.

During duplication of the genome, eukaryotic cells may experience various exogenous and endogenous replication stresses that impede progression of DNA replication along chromosomes. Chemical alterations in template DNA, imbalances of deoxynucleotide pools, repetitive sequences, tight DNA-protein complexes, and conflict with transcription can negatively affect the replication machineries. If not properly resolved, stalled replication forks can cause chromosome breaks leading to genomic instability and tumor development. Replication stress is enhanced in cancer cells due, for example, to the loss of DNA repair genes or replication-transcription conflict caused by activation of oncogenic pathways. To prevent these serious consequences, cells are equipped with diverse mechanisms that enhance the resilience of replication machineries to replication stresses. This review describes DNA damage responses activated at stressed replication forks and summarizes current knowledge on the pathways that promote faithful chromosome replication and protect chromosome integrity, including ATR-dependent replication checkpoint signaling, DNA cross-link repair, and SLX4-mediated responses to tight DNA-protein complexes that act as barriers. This review also focuses on the relevance of replication stress responses to selective cancer chemotherapies.

Proposing a molecular classification associated with hypercoagulation in ovarian clear cell carcinoma

BackgroundAlthough ovarian clear cell carcinoma (CCC) is associated with high incidence of thromboembolism, the clinicopathological and biological significance of hypercoagulable status in CCC remains unclear. Materials and methodsWe retrospectively analyzed pretreatment D-dimer levels, thromboembolic status, and clinical outcome of 125 CCCs in the discovery set and 143 CCCs in two other independent validation sets. Next, we performed RNA sequencing of 93 CCCs and compared coagulation-related gene profiles with 2492 pan-cancer data. We investigated differences in molecular characteristics of CCC subclasses based on coagulation status. ResultsIn the discovery dataset, D-dimer elevation above the normal range was significantly associated with shorter progression-free and overall survival, irrespective to thromboembolic status. Multivariate analysis identified D-dimer elevation and clinical stage as an independent prognostic factors. We confirmed the prognostic significance of D-dimer elevation in the validation sets. Tissue factor and IL6, which are considered key elements of cancer-induced hypercoagulation, were highly expressed in CCC than in other cancers regardless of D-dimer level. Higher activity of various oncogenic pathways was observed in CCC with compared to without D-dimer elevation. Moreover, hierarchical cluster analysis divided 57 CCCs with D-dimer elevation into immunologically hot and cold tumor subtypes. Hot tumors were characterized by enrichment of T-cell inflamed phenotype, inflammation, the epithelial-mesenchymal transition, and high serum levels of CRP, and cold tumors by enrichment of cell cycle and MYC pathways. ConclusionsCCC represents hypercoagulable disease and elevate D-dimer is a prognostic factor for decreased survival in CCC. D-dimer high CCC has distinct molecular characteristics into the inflammatory-driven pathway (hot tumor) and the immune-suppressive pathway (cold tumor). Treatment implication of our proposed molecular classification merits further investigation.

Altered regulation of DPF3, a member of the SWI/SNF complexes, underlies the 14q24 renal cancer susceptibility locus

SummaryOur study investigated the underlying mechanism for the 14q24 renal cell carcinoma (RCC) susceptibility risk locus identified by a genome-wide association study (GWAS). The sentinel single-nucleotide polymorphism (SNP), rs4903064, at 14q24 confers an allele-specific effect on expression of the double PHD fingers 3 (DPF3) of the BAF SWI/SNF complex as assessed by massively parallel reporter assay, confirmatory luciferase assays, and eQTL analyses. Overexpression of DPF3 in renal cell lines increases growth rates and alters chromatin accessibility and gene expression, leading to inhibition of apoptosis and activation of oncogenic pathways. siRNA interference of multiple DPF3-deregulated genes reduces growth. Our results indicate that germline variation in DPF3, a component of the BAF complex, part of the SWI/SNF complexes, can lead to reduced apoptosis and activation of the STAT3 pathway, both critical in RCC carcinogenesis. In addition, we show that altered DPF3 expression in the 14q24 RCC locus could influence the effectiveness of immunotherapy treatment for RCC by regulating tumor cytokine secretion and immune cell activation.

A Combined Bioinformatic and Nanoparticle-Based Study Reveal the Role of ABCG2 in the Drug Resistant Breast Cancer.

ATP-Binding Cassette subfamily G member 2 (ABCG2) is a semi-transport protein that plays a key role in human diseases, including bladder cancer and lung cancer, and maybe resistant to chemotherapy drugs. The present study aimed to determine the role and underlying mechanisms of breast cancer resistance protein (ABCG2) in breast cancer and to study the reversal effect of inhibiting ABCG2 expression on the drug resistance of breast cancer cells and provide new ideas for gene-targeted therapy of breast cancer. The structure and genomic alterations of ABCG2 were systematically investigated using GeneCards and cBioPortal to reveal the genetic alterations (including amplification and deep deletions) of ABCG2. We performed the correlation between ABCG2 expression and clinicopathological parameters using the data in bc-GenExMiner 4.4. Then, the protein-protein interaction and functional enrichment analysis of ABCG2 were performed based on the STRING, bc-GenExMiner 4.4, and Enrichr databases. Besides, we analyzed the pathway activity of genes that interact with ABCG2 using GSCALite and PharmGKB. Using magnetic nanoparticles polyMAG as the carrier of ABCG2-siRNA, polyMAG-ABCG2-siRNA was transfected into the Doxorubicin (DOX)-resistant breast cancer cell line MCF-7/ADR and directly into the tumors in nude mice. Patent US20150328485 points out that magnetic nanoparticles can be attached to an anti-cancer drug, such as an antibody-based anti-cancer drug. We found a statistically significant correlation between ABCG2 expression and clinicopathological parameters, such as Estrogen Receptor (ER), Progesterone Receptor (PR), and human epidermal growth factor receptor-2 (HER2), and nodal status in breast cancer patients. ABCG2 is closely related to SLC2A9, KIT, ABCG1, and MRPS7, which suggests that these proteins may be functional partners of breast cancer. The expression of ABCG2 is correlated with the activation or inhibition of multiple oncogenic pathways. Moreover, we found that ABCG2 is involved in the DOX signaling pathway. The small interfering RNA (siRNA) carried by magnetic nanoparticles can reduce the expression of ABCG2, thereby significantly improving the therapeutic effect of DOX on tumors. Our findings provide a more in-depth understanding of ABCG2 as a biomarker for predicting DOX-resistance and insights into the development of related therapeutic targets in breast cancer.

Combinatorial Strategies to Target Molecular and Signaling Pathways to Disarm Cancer Stem Cells.

Cancer is an urgent public health issue with a very huge number of cases all over the world expected to increase by 2040. Despite improved diagnosis and therapeutic protocols, it remains the main leading cause of death in the world. Cancer stem cells (CSCs) constitute a tumor subpopulation defined by ability to self-renewal and to generate the heterogeneous and differentiated cell lineages that form the tumor bulk. These cells represent a major concern in cancer treatment due to resistance to conventional protocols of radiotherapy, chemotherapy and molecular targeted therapy. In fact, although partial or complete tumor regression can be achieved in patients, these responses are often followed by cancer relapse due to the expansion of CSCs population. The aberrant activation of developmental and oncogenic signaling pathways plays a relevant role in promoting CSCs therapy resistance. Although several targeted approaches relying on monotherapy have been developed to affect these pathways, they have shown limited efficacy. Therefore, an urgent need to design alternative combinatorial strategies to replace conventional regimens exists. This review summarizes the preclinical studies which provide a proof of concept of therapeutic efficacy of combinatorial approaches targeting the CSCs.

Circadian Clock and Liver Cancer.

Simple SummaryThe circadian coordination of metabolism is tightly regulated, and its alteration can trigger several diseases, including liver steatohepatitis and cancer. Many factors (such as diet and jet lag) shape both the liver molecular clock and the circadian transcription/translation of genes related to different metabolic pathways. Here, we summarize our current knowledge about the molecular mechanisms that control this circadian regulation of liver metabolism.Circadian clocks control several homeostatic processes in mammals through internal molecular mechanisms. Chronic perturbation of circadian rhythms is associated with metabolic diseases and increased cancer risk, including liver cancer. The hepatic physiology follows a daily rhythm, driven by clock genes that control the expression of several proteins involved in distinct metabolic pathways. Alteration of the liver clock results in metabolic disorders, such as non-alcoholic fatty liver diseases (NAFLD) and impaired glucose metabolism, that can trigger the activation of oncogenic pathways, inducing spontaneous hepatocarcinoma (HCC). In this review, we provide an overview of the role of the liver clock in the metabolic and oncogenic changes that lead to HCC and discuss new potentially useful targets for prevention and management of HCC.