Dual therapeutic targeting of MYC and JUNB transcriptional programs for enhanced anti-myeloma activity.

Deregulation of transcription factors (TFs) leading to uncontrolled proliferation of tumor cells within the microenvironment represents a hallmark of cancer. However, the biological and clinical impact of transcriptional interference, particularly in multiple myeloma (MM) cells, remains poorly understood. The present study shows for the first time that MYC and JUNB, two crucial TFs implicated in MM pathogenesis, orchestrate distinct transcriptional programs. Specifically, our data revealed that expression levels of MYC, JUNB, and their respective downstream targets do not correlate and that their global chromatin-binding patterns are not significantly overlapping. Mechanistically, MYC expression was not affected by JUNB knockdown, and conversely, JUNB expression and transcriptional activity were not affected by MYC knockdown. Moreover, suppression of MYC levels in MM cells via targeting the master regulator BRD4 by either siRNA-mediated knockdown or treatment with the novel proteolysis targeting chimera (PROTAC) MZ-1 overcame bone marrow (BM) stroma cell/IL-6-induced MYC- but not MEK-dependent JUNB-upregulation and transcriptional activity. Consequently, targeting of the two non-overlapping MYC- and JUNB-transcriptoms by MZ-1 in combination with genetic or pharmacological JUNB-targeting approaches synergistically enhanced MM cell death, both in 2D and our novel dynamic 3D models of the BM milieu as well as in murine xenografts. In summary, our data emphasize the opportunity to employ MYC and JUNB dual-targeting treatment strategies in MM as another exciting approach to further improve patient outcomes.

Spatial iTME analysis of KRAS mutant NSCLC and immunotherapy outcome.

We conducted spatial immune tumor microenvironment (iTME) profiling using formalin-fixed paraffin-embedded (FFPE) samples of 25 KRAS-mutated non-small cell lung cancer (NSCLC) patients treated with immune checkpoint inhibitors (ICIs), including 12 responders and 13 non-responders. An eleven-marker panel (CD3, CD4, CD8, FOXP3, CD68, arginase-1, CD33, HLA-DR, pan-keratin (PanCK), PD-1, and PD-L1) was used to study the tumor and immune cell compositions. Spatial features at single cell level with cellular neighborhoods and fractal analysis were determined. Spatial features and different subgroups of CD68+ cells and FOXP3+ cells being associated with response or resistance to ICIs were also identified. In particular, CD68+ cells, CD33+ and FOXP3+ cells were found to be associated with resistance. Interestingly, there was also significant association between non-nuclear expression of FOXP3 being resistant to ICIs. We identified CD68dim cells in the lung cancer tissues being associated with improved responses, which should be insightful for future studies of tumor immunity.

BRD9 regulates normal human hematopoietic stem cell function and lineage differentiation.

Bromodomain containing protein 9 (BRD9), a member of the non-canonical BRG1/BRM-associated factor (ncBAF) chromatin remodeling complex, has been implicated as a synthetic lethal target in AML but its function in normal human hematopoiesis is unknown. In hematopoietic stem and progenitor cells (HSPC) genomic or chemical inhibition of BRD9 led to a proliferative disadvantage and loss of stem cells in vitro. Human HSPCs with reduced BRD9 protein levels produced lower numbers of immature mixed multipotent GEMM colonies in semi-solid media. In lineage-promoting culture conditions, cells with reduced BRD9 levels failed to differentiate into the megakaryocytic lineage and showed delayed differentiation into erythroid cells but enhanced terminal myeloid differentiation. HSPCs with BRD9 knock down (KD) had reduced long-term multilineage engraftment in a xenotransplantation assay. An increased number of downregulated genes in RNAseq analysis after BRD9 KD coupled with a gain in chromatin accessibility at the promoters of several repressive transcription factors (TF) suggest that BRD9 functions in the maintenance of active transcription during HSC differentiation. In particular, the hematopoietic master regulator GATA1 was identified as one of the core TFs regulating the gene networks modulated by BRD9 loss in HSPCs. BRD9 inhibition reduced a GATA1-luciferase reporter signal, further suggesting a role for BRD9 in regulating GATA1 activity. BRD9 is therefore an additional example of epigenetic regulation of human hematopoiesis.

The Role of Surgery in Pleural Mesothelioma.

Surgery plays a central role in the diagnosis, staging, and management of pleural mesothelioma. Achieving an accurate diagnosis through surgical intervention and identifying the specific histologic subtype is crucial for determining the appropriate course of treatment. The histologic subtype guides decisions regarding the use of chemotherapy, immunotherapy, or multimodality treatment. The goal of surgery as part of multimodality treatment is to accomplish macroscopic complete resection with the eradication of grossly visible and palpable disease. Over the past two decades, many medical centers worldwide have shifted from performing extra-pleural pneumonectomy (EPP) to pleurectomy decortication (PD). This transition is motivated by the lower rates of short-term mortality and morbidity associated with PD and similar or even better long-term survival outcomes, compared to EPP. This review aims to outline the role of surgery in diagnosing, staging, and treating patients with pleural mesothelioma.

Synergy between BRD9- and IKZF3-Targeting as a Therapeutic Strategy for Multiple Myeloma.

Progress in the treatment of multiple myeloma (MM) has resulted in improvement in the survival rate. However, there is still a need for more efficacious and tolerated therapies. We and others have shown that bromodomain-containing protein 9 (BRD9), a member of the non-canonical SWI/SNF chromatin remodeling complex, plays a role in MM cell survival, and targeting BRD9 selectively blocks MM cell proliferation and synergizes with IMiDs. We found that synergy in vitro is associated with the downregulation of MYC and Ikaros proteins, including IKZF3, and overexpression of IKZF3 or MYC could partially reverse synergy. RNA-seq analysis revealed synergy to be associated with the suppression of pathways associated with MYC and E2F target genes and pathways, including cell cycle, cell division, and DNA replication. Stimulated pathways included cell adhesion and immune and inflammatory response. Importantly, combining IMiD treatment and BRD9 targeting, which leads to the downregulation of MYC protein and upregulation of CRBN protein, was able to override IMiD resistance of cells exposed to iberdomide in long-term culture. Taken together, our results support the notion that combination therapy based on agents targeting BRD9 and IKZF3, two established dependencies in MM, represents a promising novel therapeutic strategy for MM and IMiD-resistant disease.

Chimeric Antigen Receptor T Cell Therapy in Acute Myeloid Leukemia: Trials and Tribulations.

Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy that is often associated with relapse and drug resistance after standard chemotherapy or targeted therapy, particularly in older patients. Hematopoietic stem cell transplants are looked upon as the ultimate salvage option with curative intent. Adoptive cell therapy using chimeric antigen receptors (CAR) has shown promise in B cell malignancies and is now being investigated in AML. Initial clinical trials have been disappointing in AML, and we review current strategies to improve efficacy for CAR approaches. The extensive number of clinical trials targeting different antigens likely reflects the genetic heterogeneity of AML. The limited number of patients reported in multiple early clinical studies makes it difficult to draw conclusions about CAR safety, but it does suggest that the efficacy of this approach in AML lags behind the success observed in B cell malignancies. There is a clear need not only to improve CAR design but also to identify targets in AML that show limited expression in normal myeloid lineage cells.

Acquired resistance to KRAS G12C small-molecule inhibitors via genetic/nongenetic mechanisms in lung cancer.

Inherent or acquired resistance to sotorasib poses a substantialt challenge for NSCLC treatment. Here, we demonstrate that acquired resistance to sotorasib in isogenic cells correlated with increased expression of integrin ß4 (ITGB4), a component of the focal adhesion complex. Silencing ITGB4 in tolerant cells improved sotorasib sensitivity, while overexpressing ITGB4 enhanced tolerance to sotorasib by supporting AKT-mTOR bypass signaling. Chronic treatment with sotorasib induced WNT expression and activated the WNT/ß-catenin signaling pathway. Thus, silencing both ITGB4 and ß-catenin significantly improved sotorasib sensitivity in tolerant, acquired, and inherently resistant cells. In addition, the proteasome inhibitor carfilzomib (CFZ) exhibited synergism with sotorasib by down-regulating ITGB4 and ß-catenin expression. Furthermore, adagrasib phenocopies the combination effect of sotorasib and CFZ by suppressing KRAS activity and inhibiting cell cycle progression in inherently resistant cells. Overall, our findings unveil previously unrecognized nongenetic mechanisms underlying resistance to sotorasib and propose a promising treatment strategy to overcome resistance.

A Closer Look at EGFR Inhibitor Resistance in Non-Small Cell Lung Cancer through the Lens of Precision Medicine.

The development of EGFR small-molecule inhibitors has provided significant benefit for the affected patient population. Unfortunately, current inhibitors are no curative therapy, and their development has been driven by on-target mutations that interfere with binding and thus inhibitory activity. Genomic studies have revealed that, in addition to these on-target mutations, there are also multiple off-target mechanisms of EGFR inhibitor resistance and novel therapeutics that can overcome these challenges are sought. Resistance to competitive 1st-generation and covalent 2nd- and 3rd-generation EGFR inhibitors is overall more complex than initially thought, and novel 4th-generation allosteric inhibitors are expected to suffer from a similar fate. Additional nongenetic mechanisms of resistance are significant and can include up to 50% of the escape pathways. These potential targets have gained recent interest and are usually not part of cancer panels that look for alterations in resistant patient specimen. We discuss the duality between genetic and nongenetic EGFR inhibitor drug resistance and summarize current team medicine approaches, wherein clinical developments, hand in hand with drug development research, drive potential opportunities for combination therapy.

Precision oncology provides opportunities for targeting KRAS-inhibitor resistance.

Novel inhibitors targeting Kirsten rat sarcoma virus homolog (KRAS) KRASG12C in various cancers have shown good initial efficacy, but therapy-related drug resistance eventually occurs in most patients. It has become apparent that cancer cells not only rely on novel mutations that provide escape mechanisms, but about half of them become resistant in the absence of apparent genetic mutations. Redundancies within the KRAS signaling pathways and cross-talk between these pathways - as well as other canonical cancer-driving mechanisms - not only provide challenges but also present opportunities for drug development and targeted approaches. We discuss the challenges for the duality of KRAS inhibitor drug resistance with an additional focus on nongenetic mechanisms and the potential for patient-centered combination treatments.

Clinical and Molecular Features of KRAS-Mutated Lung Cancer Patients Treated with Immune Checkpoint Inhibitors.

Background: The molecular and clinical features of KRAS-mutated lung cancer patients treated with immunotherapy have yet to be characterized, which could guide the development of therapeutics targeting KRAS with potential immuno-oncology treatment combinations. Research Question: Do KRAS-mutated patients with different subtypes and comutations have different clinical responses and overall survival (OS) to checkpoint inhibitors? Study Design and Methods: 87 patients with NSCLC at the City of Hope who received immune checkpoint inhibitors were identified and analyzed retrospectively. Tumor genomic alterations were extracted from the clinical data with next-generation sequencing using various platforms. Demographic, clinical, molecular, and pathological information was collected with the approval of the institutional review board of the City of Hope. OS was calculated if it was available at the study time point, and responses were determined according to the RECIST v1.1. Results: Among 87 patients, 32 had a KRAS G12C mutation (36.8%), 19 had G12V (21.9%), 18 had G12D (20.7%), 6 had G12A (6.9%), 3 had G12R (3.45%), and 10 had amplification (11.49%) and other uncommon mutations. G12D had a statistically significant Odds Ratio (OR) between patients who had responses and progression of the disease (OR (95% CI) = 0.31 (0.09−0.95), p < 0.05), with 5 G12D-mutated patients having responses and 11 G12D-mutated patients having progression of the disease. In the univariate analysis with OS, there was a trend of better OS in the G12D-mutated patients, with no statistically significant difference in terms of OS between the patients who had G12D mutation and the patients who had other KRAS mutations (HR (95% CI) = 0.53 (0.21−1.36), p = 0.185). The median OS was significantly worse with KRAS comutation CDKN2A/B loss (4.2 vs. 16.9 months, HR = 3.07 (1.09−8.69), p < 0.05) and MET (3.4 vs. 17 months, HR = 3.80 (1.44−10.05), p < 0.01), which were included for the multivariate analysis. The OS with other KRAS comutations was not statistically significant, including STK11 and KEAP1. Conclusion: KRAS mutation subtypes such as G12D and comutations such as CDKN2/A and MET may modulate the immunotherapy responses and outcomes in lung cancer.

BRD9 degraders as chemosensitizers in acute leukemia and multiple myeloma.

Bromodomain-containing protein 9 (BRD9), an essential component of the SWI/SNF chromatin remodeling complex termed ncBAF, has been established as a therapeutic target in a subset of sarcomas and leukemias. Here, we used novel small molecule inhibitors and degraders along with RNA interference to assess the dependency on BRD9 in the context of diverse hematological malignancies, including acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and multiple myeloma (MM) model systems. Following depletion of BRD9 protein, AML cells undergo terminal differentiation, whereas apoptosis was more prominent in ALL and MM. RNA-seq analysis of acute leukemia and MM cells revealed both unique and common signaling pathways affected by BRD9 degradation, with common pathways including those associated with regulation of inflammation, cell adhesion, DNA repair and cell cycle progression. Degradation of BRD9 potentiated the effects of several chemotherapeutic agents and targeted therapies against AML, ALL, and MM. Our findings support further development of therapeutic targeting of BRD9, alone or combined with other agents, as a novel strategy for acute leukemias and MM.

YAP1 maintains active chromatin state in head and neck squamous cell carcinomas that promotes tumorigenesis through cooperation with BRD4.

Analysis of The Cancer Genome Atlas and other published data of head and neck squamous cell carcinoma (HNSCC) reveals somatic alterations of the Hippo-YAP pathway in approximately 50% of HNSCC. Better strategies to target the YAP1 transcriptional complex are sought. Here, we show that FAT1, an upstream inhibitor of YAP1, is mutated either by missense or by truncating mutation in 29% of HNSCC. Comprehensive proteomic and drug-screening studies across pan-cancer models confirm that FAT1-mutant HNSCC exhibits selective and higher sensitivity to BRD4 inhibition by JQ1. Epigenomic analysis reveals an active chromatin state in FAT1-mutant HNSCC cells that is driven by the YAP/TAZ transcriptional complex through recruitment of BRD4 to deposit active histone marks, thereby maintaining an oncogenic transcriptional state. This study reveals a detailed cooperative mechanism between YAP1 and BRD4 in HNSCC and suggests a specific therapeutic opportunity for the treatment of this subset of head and neck cancer patients.

Small molecule inhibition of deubiquitinating enzyme JOSD1 as a novel targeted therapy for leukemias with mutant JAK2.

Mutations in the Janus Kinase 2 (JAK2) gene resulting in constitutive kinase activation represent the most common genetic event in myeloproliferative neoplasms (MPN), a group of diseases involving overproduction of one or more kinds of blood cells, including red cells, white cells, and platelets. JAK2 kinase inhibitors, such as ruxolitinib, provide clinical benefit, but inhibition of wild-type (wt) JAK2 limits their clinical utility due to toxicity to normal cells, and small molecule inhibition of mutated JAK2 kinase activity can lead to drug resistance. Here, we present a strategy to target mutated JAK2 for degradation, using the cell's intracellular degradation machinery, while sparing non-mutated JAK2. We employed a chemical genetics screen, followed by extensive selectivity profiling and genetic studies, to identify the deubiquitinase (DUB), JOSD1, as a novel regulator of mutant JAK2. JOSD1 interacts with and stabilizes JAK2-V617F, and inactivation of the DUB leads to JAK2-V617F protein degradation by increasing its ubiquitination levels, thereby shortening its protein half-life. Moreover, targeting of JOSD1 leads to the death of JAK2-V617F-positive primary acute myeloid leukemia (AML) cells. These studies provide a novel therapeutic approach to achieving selective targeting of mutated JAK2 signaling in MPN.

Protein Phosphatase 2A as a Therapeutic Target in Small Cell Lung Cancer.

Protein phosphatase 2A (PP2A), a serine/threonine phosphatase involved in the regulation of apoptosis, proliferation, and DNA-damage response, is overexpressed in many cancers, including small cell lung cancer (SCLC). Here we report that LB100, a small molecule inhibitor of PP2A, when combined with platinum-based chemotherapy, synergistically elicited an antitumor response both in vitro and in vivo with no apparent toxicity. Using inductively coupled plasma mass spectrometry, we determined quantitatively that sensitization via LB100 was mediated by increased uptake of carboplatin in SCLC cells. Treatment with LB100 alone or in combination resulted in inhibition of cell viability in two-dimensional culture and three-dimensional spheroid models of SCLC, reduced glucose uptake, and attenuated mitochondrial and glycolytic ATP production. Combining LB100 with atezolizumab increased the capacity of T cells to infiltrate and kill tumor spheroids, and combining LB100 with carboplatin caused hyperphosphorylation of the DNA repair marker γH2AX and enhanced apoptosis while attenuating MET signaling and invasion through an endothelial cell monolayer. Taken together, these data highlight the translational potential of inhibiting PP2A with LB100 in combination with platinum-based chemotherapy and immunotherapy in SCLC.

Essential role of the histone lysine demethylase KDM4A in the biology of malignant pleural mesothelioma (MPM).

BACKGROUND: Malignant pleural mesothelioma (MPM) is a highly aggressive cancer with a dismal prognosis. There is increasing interest in targeting chromatin regulatory pathways in difficult-to-treat cancers. In preliminary studies, we found that KDM4A (lysine-specific histone demethylase 4) was overexpressed in MPM. METHODS: KDM4A protein expression was determined by immunohistochemistry or immunoblotting. Functional inhibition of KDM4A by targeted knockdown and small molecule drugs was correlated to cell growth using cell lines and a xenograft mouse model. Gene expression profiling was performed to identify KDM4A-dependent signature pathways. RESULTS: Levels of KDM4A were found to be significantly elevated in MPM patients compared to normal mesothelial tissue. Inhibiting the enzyme activity efficiently reduced cell growth in vitro and reduced tumour growth in vivo. KDM4A inhibitor-induced apoptosis was further enhanced by the BH3 mimetic navitoclax. KDM4A expression was associated with pathways involved in cell growth and DNA repair. Interestingly, inhibitors of the DNA damage and replication checkpoint regulators CHK1 (prexasertib) and WEE1 (adavosertib) within the DNA double-strand break repair pathway, cooperated in the inhibition of cell growth. CONCLUSIONS: The results establish a novel and essential role for KDM4A in growth in preclinical models of MPM and identify potential therapeutic approaches to target KDM4A-dependent vulnerabilities.