Bromodomain And Extra-terminal Motif Research Articles

A phenotypic screening approach to target p60AmotL2-expressing invasive cancer cells

BackgroundTumor cells have the ability to invade and form small clusters that protrude into adjacent tissues, a phenomenon that is frequently observed at the periphery of a tumor as it expands into healthy tissues. The presence of these clusters is linked to poor prognosis and has proven challenging to treat using conventional therapies. We previously reported that p60AmotL2 expression is localized to invasive colon and breast cancer cells. In vitro, p60AmotL2 promotes epithelial cell invasion by negatively impacting E-cadherin/AmotL2-related mechanotransduction.MethodsUsing epithelial cells transfected with inducible p60AmotL2, we employed a phenotypic drug screening approach to find compounds that specifically target invasive cells. The phenotypic screen was performed by treating cells for 72 h with a library of compounds with known antitumor activities in a dose-dependent manner. After assessing cell viability using CellTiter-Glo, drug sensitivity scores for each compound were calculated. Candidate hit compounds with a higher drug sensitivity score for p60AmotL2-expressing cells were then validated on lung and colon cell models, both in 2D and in 3D, and on colon cancer patient-derived organoids. Nascent RNA sequencing was performed after BET inhibition to analyse BET-dependent pathways in p60AmotL2-expressing cells.ResultsWe identified 60 compounds that selectively targeted p60AmotL2-expressing cells. Intriguingly, these compounds were classified into two major categories: Epidermal Growth Factor Receptor (EGFR) inhibitors and Bromodomain and Extra-Terminal motif (BET) inhibitors. The latter consistently demonstrated antitumor activity in human cancer cell models, as well as in organoids derived from colon cancer patients. BET inhibition led to a shift towards the upregulation of pro-apoptotic pathways specifically in p60AmotL2-expressing cells.ConclusionsBET inhibitors specifically target p60AmotL2-expressing invasive cancer cells, likely by exploiting differences in chromatin accessibility, leading to cell death. Additionally, our findings support the use of this phenotypic strategy to discover novel compounds that can exploit vulnerabilities and specifically target invasive cancer cells.

BET-inhibitor DYB-41 reduces pulmonary inflammation and local and systemic cytokine levels in LPS-induced acute respiratory distress syndrome: an experimental rodent study

BackgroundAcute respiratory distress syndrome (ARDS) is a form of respiratory failure stemming from various underlying conditions that ultimately lead to inflammation and lung fibrosis. Bromodomain and Extra-Terminal motif (BET) inhibitors are a class of medications that selectively bind to the bromodomains of BET motif proteins, effectively reducing inflammation. However, the use of BET inhibitors in ARDS treatment has not been previously investigated. In our study, we induced ARDS in rats using endotoxin and administered a BET inhibitor. We evaluated the outcomes by examining inflammation markers and lung histopathology.ResultsNine animals received treatment, while 12 served as controls. In the lung tissue of treated animals, we observed a significant reduction in TNFα levels (549 [149–977] pg/mg vs. 3010 [396–5529] pg/mg; p = 0.009) and IL-1β levels (447 [369–580] pg/mg vs. 662 [523–924] pg/mg; p = 0.012), although IL-6 and IL-10 levels showed no significant differences. In the blood, treated animals exhibited a reduced TNFα level (25 [25–424] pg/ml vs. 900 [285–1744] pg/ml, p = 0.016), but IL-1β levels were significantly higher (1254 [435–2474] pg/ml vs. 384 [213–907] pg/ml, p = 0.049). No differences were observed in IL-6 and IL-10 levels. There were no significant variations in lung tissue levels of TGF-β, SP-D, or RAGE. Histopathological analysis revealed substantial damage, with notably less perivascular edema (3 vs 2; p = 0.0046) and visually more inflammatory cells. However, two semi-quantitative histopathologic scoring systems did not indicate significant differences.ConclusionsThese preliminary findings suggest a potential beneficial effect of BET inhibitors in the treatment of acute lung injury and ARDS. Further validation and replication of these results with a larger cohort of animals, in diverse models, and using different BET inhibitors are needed to explore their clinical implications.

BET inhibition induces synthetic lethality in PTEN deficient colorectal cancers via dual action on p21CIP1/WAF1.

Loss of PTEN tumor suppressor is an important event during colorectal cancer (CRC) development and is a target for therapeutic exploitation. This study reports that bromodomain and extra-terminal motif (BET) is a synthetic lethal partner of PTEN in CRC. BET inhibition (BETi) selectively induced G1 cell cycle arrest and apoptosis in PTEN-/- CRC. Further, BETi selectively and dose-dependently suppressed the growth of PTEN-/- CRC tumor xenografts in mice and patient-derived organoids. Mechanistically, PTEN-deficient CRC cells elevated the level of cytoplasmic p21CIP1/WAF1 that is hyper-phosphorylated at Thr145 by AKT. BETi suppressed AKT activation in PTEN-deficient CRC cells, followed by the reduction in p21 phosphorylation at Thr145, thereby promoting its nuclear translocation. In addition, BETi suppressed MYC level and this in turn increased the total p21 level in the nuclei. Over-expression of a phospho-mimetic p21 mutant (T145D) significantly rescued the BETi effect on PTEN-deficient CRC. These results suggest that BETi has a dual action on p21: elevating the level of p21 by inhibiting MYC and converting the oncogenic (cytoplasmic) p21 into the tumor-suppressive (nuclear) p21 by inhibiting AKT. Taken together, this study identified the synthetic lethal interaction between PTEN and BET, and provides a potential actionable target for CRC with PTEN loss.

Abstract C109: INTASYL™ PH-894 self-delivering RNAi targeting BRD4 enhances the antigenicity of melanoma cells through MART-1 upregulation

Abstract Background: BRD4 is a member of the family of bromodomain and extra-terminal motif (BET) proteins that act as epigenetic readers and regulators of gene transcription. BRD4 has been shown to be significantly upregulated in cancer including melanoma, with preclinical data supporting BRD4 inhibition as a promising therapeutic target. However, clinical translation of BRD4 inhibitors (BRD4i) has remained elusive. Current small molecule inhibitors of BET proteins are not specific to BRD4 as demonstrated by their inhibition of other members of the BET protein family. Additionally, systemic approaches must overcome a narrow therapeutic window. The INTASYL™ platform is a self-delivering RNAi technology that imparts small molecule-like properties to interfering RNAs, providing efficient delivery to target cells without need for specialized formulations or drug delivery systems. PH-894 is an INTASYL compound that silences BRD4 mRNA. PH-894 has been shown to specifically silence BRD4 mRNA and provide robust direct and abscopal antitumor efficacy in vivo when administered intratumorally (IT). Therefore, IT PH-894 holds promise to realize the therapeutic potential of BRD4 inhibition by avoiding toxicities associated with systemic delivery of small molecule BET inhibitors. Here, we explored the impact of PH-894 mediated BRD4 inhibition on antigenicity of melanoma tumor cells. Specifically, expression of the melanoma tumor-associated antigen MART-1 was studied following PH-894 treatment. Methods: SK-MEL-5 melanoma cells were treated for 72 hours with PH-894 or a non-targeting control (NTC) INTASYL compound. After 72 hours (Day 3), PH-894 was washed out and cells were replated in media containing no INTAYSL compound for the duration of the experiment. Cells were collected for analysis of both BRD4 and MART-1 mRNA and protein from Day 3 through Day 7. Results: PH-894 provided robust silencing of BRD4 mRNA and protein in SK-MEL-5 melanoma cells. Furthermore, PH-894 treatment resulted in a statistically significant upregulation of MART-1 mRNA and protein in these cells. BRD4 silencing and MART-1 upregulation persisted through at least 7 days post-treatment with PH-894. Conclusions: This study demonstrates the ability of PH-894 to efficiently silence BRD4 and upregulate MART-1, thereby increasing the antigenicity of melanoma cells. Local treatment with PH-894 is a potential strategy to decrease BRD4 expression and increase immune response to cancer cells while reducing toxicities associated with systemic BETi. This study supports further development of PH-894 for injectable solid tumor indications such as melanoma. Citation Format: Brianna Rivest, Melissa Maxwell, Andrew Boone, Benjamin Cuiffo, Dingxue Yan, James Cardia. INTASYL™ PH-894 self-delivering RNAi targeting BRD4 enhances the antigenicity of melanoma cells through MART-1 upregulation [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C109.

Abstract C107: INTASYL™ self-delivering RNAi therapeutic targeting BRD4 elicits on-target apoptosis of human tumor cells

Abstract Background: A wealth of preclinical data supports the therapeutic potential of targeting bromodomain and extra-terminal motif (BET) protein BRD4 as a promising strategy to treat a spectrum of cancers. However, clinical translation of BRD4 inhibitors (BRD4i) has not yet been achieved. While clinical trials for next generation small molecule BRD4i compounds are ongoing, such systemic approaches must overcome a narrow therapeutic window. Therefore, a local approach to BRD4i represents a potential strategy to realize its therapeutic potential in injectable solid tumor indications. INTASYL™ is a self-delivering RNAi platform technology that enables rapid and efficient delivery into target cells without need for specialized drug delivery systems or formulations. INTASYL compounds provide precise and durable gene silencing. In preclinical studies, INTASYL compounds provided a robust direct, abscopal and/or anti-metastatic antitumor efficacy when administered intratumorally (IT) as monotherapy, multi-targeting formulations, or in combination with current standard of care treatments. PH-894 is an INTASYL compound designed to specifically silence human BRD4 mRNA. We assessed the direct on-target antitumor effects of PH-894 across a spectrum of human tumor cell lines: HepG2 (hepatocellular carcinoma), HCC1806 (acantholytic squamous cell carcinoma), A549 (lung adenocarcinoma), U-251 MG (malignant glioblastoma), SK-MEL-5 (melanoma), HT-29 (colorectal adenocarcinoma), SK-OV-3 (ovarian adenocarcinoma), HeLa (cervical adenocarcinoma). Methods: Tumor cells were cell-cycle synchronized by serum starvation for ~18 h and treated in the presence of serum with increasing concentrations of PH-894 or a non-targeting control (NTC) INTASYL for 24 h. Apoptosis was assessed by annexin-v and 7-aminoactinomycin D (7AAD) staining/flow cytometry. On-target silencing of BRD4 mRNA by PH-894 was confirmed at 24 h by qPCR. Results: PH-894 elicited on-target apoptosis in all tumor cell lines tested in a concentration correlated manner, with variable levels of response across the various tumor cell lines. BRD4 mRNA was specifically silenced on-target in a concentration correlated manner with PH-894. Conclusions: This study demonstrates the direct cytotoxic effects of INTASYL PH-894 mediated by sequence specific silencing of BRD4 mRNA across tumor cell lines representing a broad spectrum of injectable solid tumors. Local therapy with IT PH-894 represents a potential strategy to mitigate toxicities associated with systemic BETi or BRD4i. This study supports further development of PH-894 toward unlocking the therapeutic potential of BRD4i. Citation Format: Andrew Boone, Dingxue Yan, Melissa Maxwell, Brianna Rivest, James Cardia, Benjamin Cuiffo. INTASYL™ self-delivering RNAi therapeutic targeting BRD4 elicits on-target apoptosis of human tumor cells [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C107.

P11 - Bromodomain and Extra-Terminal motif (BET) inhibitors sensitize bladder carcinoma cells to PARP inhibitors by inducing DNA repair deficiency (BRCAness)

P11 - Bromodomain and Extra-Terminal motif (BET) inhibitors sensitize bladder carcinoma cells to PARP inhibitors by inducing DNA repair deficiency (BRCAness)

A new vulnerability to BET inhibition due to enhanced autophagy in BRCA2 deficient pancreatic cancer

Pancreatic cancer is one of the deadliest diseases in human malignancies. Among total pancreatic cancer patients, ~10% of patients are categorized as familial pancreatic cancer (FPC) patients, carrying germline mutations of the genes involved in DNA repair pathways (e.g., BRCA2). Personalized medicine approaches tailored toward patients’ mutations would improve patients’ outcome. To identify novel vulnerabilities of BRCA2-deficient pancreatic cancer, we generated isogenic Brca2-deficient murine pancreatic cancer cell lines and performed high-throughput drug screens. High-throughput drug screening revealed that Brca2-deficient cells are sensitive to Bromodomain and Extraterminal Motif (BET) inhibitors, suggesting that BET inhibition might be a potential therapeutic approach. We found that BRCA2 deficiency increased autophagic flux, which was further enhanced by BET inhibition in Brca2-deficient pancreatic cancer cells, resulting in autophagy-dependent cell death. Our data suggests that BET inhibition can be a novel therapeutic strategy for BRCA2-deficient pancreatic cancer.

BET inhibition targets ABC-DLBCL constitutive B-cell receptor signaling through PAX5.

B-cell receptor (BCR) signaling is essential for the diffuse large B-cell lymphoma (DLBCL) subtype that originates from activated B-cells (ABCs). ABC-DLBCL cells are sensitive to Bruton tyrosine kinase intervention. However, patients with relapsed or refractory ABC-DLBCL had overall response rates from 33% to 37% for Bruton tyrosine kinase inhibitors, suggesting the evaluation of combination-based treatment for improved efficacy. We investigated the efficacy and mechanism of the bromodomain and extraterminal motif (BET) inhibitor AZD5153 combined with the Bruton tyrosine kinase inhibitor acalabrutinib in ABC-DLBCL preclinical models. AZD5153 is a bivalent BET inhibitor that simultaneously engages the 2 bromodomains of BRD4. Adding AZD5153 to acalabrutinib demonstrated combination benefits in ABC-DLBCL cell line and patient-derived xenograft models. Differential expression analyses revealed PAX5 transcriptional activity as a novel downstream effector of this drug combination. PAX5 is a transcription factor for BCR signaling genes and may be critical for perpetually active BCR signaling in ABC-DLBCL. Our analyses further indicated significant alterations in BCR, RELB/alternative NF-κB, and toll-like receptor/interferon signaling. Validation of these results mapped a positive-feedback signaling loop regulated by PAX5. We demonstrated that AZD5153 decreased PAX5 expression, whereas acalabrutinib disruption of BCR signaling inhibited PAX5 activation. Furthermore, several interferon levels were decreased by AZD5153 and acalabrutinib in tumors. Adding interferon-beta1 (IFNβ1) to cells treated with acalabrutinib partially rescued PAX5 activation. Our results demonstrate that AZD5153 enhances the efficacy of acalabrutinib through PAX5 and BCR mechanisms that are critical for ABC-DLBCL.

Streamlined DNA-encoded small molecule library screening and validation for the discovery of novel chemotypes targeting BET proteins

Targeting aberrant epigenetic programs that drive tumorigenesis is a promising approach to cancer therapy. DNA-encoded library (DEL) screening is a core platform technology increasingly used to identify drugs that bind to protein targets. Here, we use DEL screening against bromodomain and extra-terminal motif (BET) proteins to identify inhibitors with new chemotypes, and successfully identified BBC1115 as a selective BET inhibitor. While BBC1115 does not structurally resemble OTX-015, a clinically active pan-BET inhibitor, our intensive biological characterization revealed that BBC1115 binds to BET proteins, including BRD4, and suppresses aberrant cell fate programs. Phenotypically, BBC1115-mediated BET inhibition impaired proliferation in acute myeloid leukemia, pancreatic, colorectal, and ovarian cancer cells invitro. Moreover, intravenous administration of BBC1115 inhibited subcutaneous tumor xenograft growth with minimal toxicity and favorable pharmacokinetic properties invivo. Since epigenetic regulations are ubiquitously distributed across normal and malignant cells, it will be critical to evaluate if BBC1115 affects normal cell function. Nonetheless, our study shows integrating DEL-based small-molecule compound screening and multi-step biological validation represents a reliable strategy to discover new chemotypes with selectivity, efficacy, and safety profiles for targeting proteins involved in epigenetic regulation in human malignancies.

Targeting PKLR/MYCN/ROMO1 signaling suppresses neuroendocrine differentiation of castration-resistant prostate cancer

Conventional treatment of prostate cancer (PCa) uses androgen-deprivation therapy (ADT) to inhibit androgen receptor (AR) signaling-driven tumor progression. ADT-induced PCa recurrence may progress to an AR-negative phenotype with neuroendocrine (NE) histologic features, which are associated with metabolic disturbances and poor prognoses. However, the metabolic pathways that regulate NE differentiation (NED) in PCa remain unclear. Herein, we show a regulatory mechanism in NED-associated metabolism dysfunction induced by ADT, whereby overexpression of pyruvate kinase L/R (PKLR) mediates oxidative stress through upregulation of reactive oxygen species modulator 1 (ROMO1), thereby promoting NED and aggressiveness. ADT mediates the nuclear translocation of PKLR, which binds to the MYCN/MAX complex to upregulate ROMO1 and NE-related genes, leading to altered mitochondrial function and NED of PCa. Targeting nuclear PKLR/MYCN using bromodomain and extra-terminal motif (BET) inhibitors has the potential to reduce PKLR/MYCN-driven NED. Abundant ROMO1 in serum samples may provide prognostic information in patients with ADT. Our results suggest that ADT resistance leads to upregulation of PKLR/MYCN/ROMO1 signaling, which may drive metabolic reprogramming and NED in PCa. We further show that increased abundance of serum ROMO1 may be associated with the development of NE-like PCa.

Reset of Inflammatory Priming of Joint Tissue and Reduction of the Severity of Arthritis Flares by Bromodomain Inhibition.

We have recently shown that priming of synovial fibroblasts (SFs) drives arthritis flares. Pathogenic priming of SFs is essentially mediated by epigenetic reprogramming. Bromodomain and extraterminal motif (BET) proteins translate epigenetic changes into transcription. Here, we used a BET inhibitor (I-BET151) to target inflammatory tissue priming and to reduce flare severity in a murine experimental arthritis model. BALB/c mice were treated by intraperitoneal injection or by local injection in the paw with I-BET151, which blocks the interaction of BET proteins with acetylated histones. We assessed the effects of I-BET151 on acute arthritis and/or inflammatory tissue priming in a model of repeated injections of monosodium urate crystals or zymosan into the mouse paw. I-BET151 was given before arthritis induction, at peak inflammation, or after healing of the first arthritis bout. We performed transcriptomic (RNA-Seq), epigenomic (ATAC-Seq), and functional (invasion, cytokine production, migration, senescence, metabolic flux) analyses of murine and human SFs treated with I-BET151 in vitro or in vivo. Systemic I-BET151 administration did not affect acute inflammation but abolished inflammatory tissue priming and diminished flare severity in both preventive and therapeutic treatment settings. I-BET151 was also effective when applied locally in the joint. BET inhibition also inhibited osteoclast differentiation, while macrophage activation in the joint was not affected. Flare reduction after BET inhibition was mediated, at least in part, by rolling back the primed transcriptional, metabolic, and pathogenic phenotype of SFs. Inflammatory tissue priming is dependent on transcriptional regulation by BET proteins, making them promising therapeutic targets for prevention of arthritis flares in previously affected joints.

Abstract B014: Mutant p53-mediated CSF1/CSF1R signaling promotes tumor invasion and lung metastasis in esophageal squamous cell carcinoma

Abstract Introduction: Mutations of the tumor suppressor p53 are detected up to 80% of esophageal squamous cell carcinoma (ESCC) cases, which in turn correlate with high metastatic rates and poor prognosis. To understand the mutant p53-mediated mechanisms in promoting ESCC metastasis, we conducted RNA-Seq and cytokine array on isogenic primary and metastatic tumor cells harvested from our mouse model of esophageal cancer harboring Trp53R172H/-, in which we have identified Colony stimulating factor 1 (Csf1) to be upregulated. Our goal is to investigate the role and mediators of CSF1 signaling through its cognate receptor CSF1R by which missense p53 mutations can promote tumor invasion and lung metastasis in ESCC. Methods: We have utilized novel L2-Cre; LSL-Trp53R172H; Rosa26LSL-YFP mice and isolated tumor cells to model metastatic ESCC. We conducted ChIP-Seq analysis for p53 on tumor cells derived from lung metastases in our mouse models. Furthermore, we have genetically and pharmacologically targeted CSF1/CSF1R signaling axis to assess its role in ESCC tumor invasion and lung metastasis. In addition to studying tumor intrinsic mechanisms, we have established quantitative multiplex immunofluorescence (qmIF) and flow cytometry approaches to characterize the changes in tumor microenvironment (TME). Results And Discussion: We demonstrate that metastatic ESCC has increased Csf1 expression compared to primary tumors, and this is dependent upon p53 mutation status, which is reinforced by the TCGA data and patient-derived tissue microarrays (TMAs). The overlay of the RNA-Seq with the ChIP-Seq analysis indicates that Csf1 is a direct gene target of p53-R172H with enriched binding motifs. Furthermore, based on the proximity ligation assay (PLA) with murine ESCC tumor cells, bromodomain and extra-terminal motif (BET) protein BRD4 interacts with p53-R172H to induce Csf1 expression. We show that the BRD4-CSF1 axis fosters tumor invasion, subcutaneous tumor growth and metastatic burden in a mutant p53 background. In accordance, upon inhibiting this signaling pathway in the tail-vein injection lung metastasis models, we have identified reduced expression of CD31+ cells and decreased infiltration of F4/80+CD163+ and F4/80+CD206+ M2-polarized macrophages at the metastatic tumor sites, indicating that the CSF1/CSF1R pathway plays a critical role in shaping the pro-metastatic and immunosuppressive TME. Finally, analysis of the esophageal cancer datasets reveals that specific p53 mutations are associated with markedly differential overall survival rates and Csf1 expression. Based upon these results, we are generating human and murine esophageal cells with distinct DNA contact and conformational p53 mutations via base editing. Conclusion: We have demonstrated novel roles and mechanisms of mutant p53-dependent CSF1/CSF1R signaling pathway in fostering ESCC tumor invasion and lung metastasis that may be applicable to other squamous cell cancers. We believe this can open up new avenues for therapeutic applications. Citation Format: Gizem Efe, Qiaosi Tang, Katherine M. Cunningham, Kensuke Sugiura, Karen Dunbar, Kausik Regunath, Kensuke Suzuki, Andres J. Klein-Szanto, Lois Resnick-Silverman, James J. Manfredi, Carol L. Prives, Anil K. Rustgi. Mutant p53-mediated CSF1/CSF1R signaling promotes tumor invasion and lung metastasis in esophageal squamous cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B014.

The Combination of Navitoclax and Ruxolitinib in JAK Inhibitor-Naïve Patients with Myelofibrosis Mediates Responses Suggestive of Disease Modification

Background: Myelofibrosis (MF) is a myeloproliferative neoplasm associated with splenomegaly, high symptom burden, bone marrow fibrosis (BMF), and poor prognosis. The Janus kinase inhibitor (JAKi) ruxolitinib is the current standard of care for patients with MF. It improves splenomegaly and disease symptoms with limited impact on disease biology, highlighting an unmet medical need in MF. Driver mutations of JAK2, CALR, and MPL are key to the pathogenesis of MF. Reduction in variant allele frequency (VAF) of driver mutations and reversal of BMF are suggestive of disease modification (Pemmaraju, Cancer, 2022). Additionally, mutations in ASXL1, EZH2, SRSF2, IDH1/2, and U2AF1 p.Q157 are classified as high molecular risk (HMR) mutations associated with a worse prognosis (Arber et al., Blood, 2016). We previously demonstrated evidence of disease modification independent of HMR mutations by adding navitoclax to ruxolitinib in patients with suboptimal responses to ruxolitinib monotherapy (Pemmaraju, Lancet Oncology, 2022). Herein, we investigated whether the type of MF and risk (e.g., age, Dynamic International Prognostic Scoring System [DIPSS], and HMR) correlated with clinical outcomes and responses suggestive of disease modification (e.g., VAF reduction and improvement in BMF) among JAKi treatment-naïve patients with MF treated with the combination of navitoclax and ruxolitinib. Methods: Cohort-3 of the phase-2 multicenter REFINE study (NCT03222609) enrolled JAKi treatment-naïve patients with MF. Enrolled patients had primary or secondary MF with splenomegaly (DIPSS ≥ Intermediate-1) and did not receive prior JAK-2 therapy or bromodomain and extra terminal motif (BET) inhibitors. Patients initiated navitoclax at 100 mg QD or 200 mg QD if baseline platelet count was ≤ 150 × 109/L or > 150 × 109/L, respectively. Ruxolitinib was given BID with starting dose based on baseline platelet count per local label. The primary endpoint was spleen volume reduction of ≥ 35% (SVR35) from baseline at week 24, assessed by MRI conducted by central review. Key secondary and exploratory endpoints evaluated in this analysis were a reduction in BMF obtained from BM biopsies by local evaluation and a reduction in VAF for the driver gene mutations (JAK2V617, CALR, or MPL), respectively. Driver gene VAF and HMR mutations were determined in whole blood with the 50-gene Focus Myeloid next-generation sequencing panel (Interspace Pharma Solutions, Morrisville, NC, USA). Results: As of Feb 07, 2022, all enrolled patients (N = 32) treated with navitoclax and ruxolitinib were evaluable for biomarker analysis. The median duration of follow-up was 6.1 months (range, 1.9 – 18.6); median age was 69 years (range, 44 – 83), and median spleen volume was 1889.08 cm3 (range, 645.6 – 7339.6). At baseline, 22 (69%) patients had JAK2, 6 (19%) had CALR, and 3 (9%) had MPL mutations. One (3%) patient was triple negative. Nineteen (59%) patients had HMR mutations. SVR35 at week 24 was observed in all subgroups known to confer poor prognosis, including age (≥75 years, 50% [n = 4/8]), high DIPSS score (Intermediate-2, 63% [n = 12/19]; high, 33% [n = 1/3]), and HMR mutations (47% [n = 9/19]; Figure). BMF grade improvement was evaluable in 26/32 (81%) patients, of which 9/26 (35%) achieved ≥ 1-grade improvement at any time during treatment with a median time-to-improvement of 12.3 weeks (range, 12.1 – 24.1). Complete resolution of BMF was observed in 2/9 (22%) patients. The baseline BMF grades of these patients were 2 and 3. BMF ≥ 1-grade improvement was achieved by 7/13 (54%) patients with HMR mutations and 2/13 (15%) without HMR mutations. Reduction in driver gene JAK2V617 mutation VAF > 20% from baseline at week 12 or 24 was observed in 14/28 (50%) patients, and 5/14 (36%) patients achieved > 50% VAF reduction from baseline. There were no differences in > 20% VAF reductions from baseline to week 12 or 24 between those with or without HMR mutations (7/15 [47%] versus 7/13 [54%], respectively). Conclusions: Among JAKi treatment-naïve patients with MF, the combination of navitoclax and ruxolitinib reduced splenomegaly in several high-risk groups known to confer poor prognosis. Reductions in BMF and VAF were independent of HMR mutations. The reduction in BMF and VAF for the driver mutation JAK2V617 is encouraging and suggestive of evidence of disease modification with the combination of navitoclax and ruxolitinib. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Genome-wide CRISPR screens identify combinations of candidate latency reversing agents for targeting the latent HIV-1 reservoir.

Reversing HIV-1 latency promotes killing of infected cells and is essential for cure strategies; however, no single latency reversing agent (LRA) or LRA combination have been shown to reduce HIV-1 latent reservoir size in persons living with HIV-1 (PLWH). Here, we describe an approach to systematically identify LRA combinations to reactivate latent HIV-1 using genome-wide CRISPR screens. Screens on cells treated with suboptimal concentrations of an LRA can identify host genes whose knockout enhances viral gene expression. Therefore, inhibitors of these genes should synergize with the LRA. We tested this approach using AZD5582, an activator of the noncanonical nuclear factor κB (ncNF-κB) pathway, as an LRA and identified histone deacetylase 2 (HDAC2) and bromodomain-containing protein 2 (BRD2), part of the bromodomain and extra-terminal motif (BET) protein family targeted by BET inhibitors, as potential targets. Using CD4+ T cells from PLWH, we confirmed synergy between AZD5582 and several HDAC inhibitors and between AZD5582 and the BET inhibitor, JQ1. A reciprocal screen using suboptimal concentrations of an HDAC inhibitor as an LRA identified BRD2 and ncNF-κB regulators, especially BIRC2, as synergistic candidates for use in combination with HDAC inhibition. Moreover, we identified and validated additional synergistic drug candidates in latency cell line cells and primary lymphocytes isolated from PLWH. Specifically, the knockout of genes encoding CYLD or YPEL5 displayed synergy with existing LRAs in inducing HIV mRNAs. Our study provides insights into the roles of host factors in HIV-1 reactivation and validates a system for identifying drug combinations for HIV-1 latency reversal.

NUT Carcinoma-An Underdiagnosed Malignancy.

NUT carcinoma (NC) is a rare and highly aggressive malignancy with a dismal prognosis and a median survival of 6–9 months only. Although very few cases of NC are reported each year, the true prevalence is estimated to be much higher, with NC potentially widely underdiagnosed due to the lack of awareness. NC primarily occurs in midline structures including thorax, head, and neck; however, other sites such as pancreas and kidney are also affected, albeit at lower frequencies. NC is characterized by a single translocation involving the NUTM1 (NUT midline carcinoma family member 1) gene and different partner genes. The resulting fusion proteins initiate tumorigenesis through a mechanism involving BET (bromo-domain and extra-terminal motif) proteins such as Bromodomain-containing protein 4 (BRD4) and inordinate acetylation of chromatin, leading to the dysregulation of growth and differentiation genes. While no clinical characteristics are specific for NC, some histologic features can be indicative; therefore, patients with these tumor characteristics should be routinely tested for NUTM1. The diagnosis of NC using immunohistochemistry with a highly specific antibody is straightforward. There are currently no standard-of-care treatment options for patients with NC. However, novel therapies specifically addressing the unique tumorigenic mechanism are under investigation, including BET inhibitors. This review aims to raise awareness of this underdiagnosed cancer entity and provide all patients the opportunity to be properly diagnosed and referred to a clinical study.

S197: NAVITOCLAX PLUS RUXOLITINIB IN JAK INHIBITOR-NAÏVE PATIENTS WITH MYELOFIBROSIS: PRELIMINARY SAFETY AND EFFICACY IN A MULTICENTER, OPEN-LABEL PHASE 2 STUDY

Background: Ruxolitinib, a Janus kinase (JAK) 1/2 inhibitor, is the current standard of care for patients with myelofibrosis (MF) that improves disease symptoms in approximately 40% of patients with limited impact on disease biology. Many patients lose response over time, highlighting an unmet need for novel therapies. Navitoclax is a first-in-class, oral, small molecule that binds with high affinity to BCL-XL and BCL-2, key pro-survival proteins in the apoptotic pathway, and has a synergistic effect when used in combination with JAK inhibitors to enhance malignant cell death in MF. This ongoing, open-label, multicenter, phase II trial (NCT03222609) is evaluating the efficacy and safety of navitoclax with or without ruxolitinib in patients with MF and previously published data suggest clinically meaningful activity of the combination in patients with prior ruxolitinib failure (Pemmaraju et al., ASH 2020). Here, we report results from patients who were JAK inhibitor-naïve and treated with navitoclax and ruxolitinib combination therapy. Aims: N/A Methods: Enrolled patients had primary or secondary MF with splenomegaly (DIPSS ≥ Intermediate-1) and did not receive prior therapy with JAK-2 or bromodomain and extraterminal motif (BET) inhibitors. Patients initiated navitoclax at 100 mg once per day (QD) or 200 mg QD if baseline platelet count was ≤150 × 109/L or >150 × 109/L, respectively. Ruxolitinib was orally administered twice daily at a starting dose based on baseline platelet count as per the local ruxolitinib label. The primary endpoint was spleen volume reduction of ≥35% (SVR35) from baseline at week 24. SVR35 was assessed at weeks 12, 24, and once every 24 weeks thereafter. Key secondary endpoints were ≥50% reduction in total symptom score (TSS50), bone marrow fibrosis reduction, and anemia response (per International Working Group criteria). Adverse events (AEs) were monitored throughout the study. Results: As of Oct 04, 2021, 32 patients received navitoclax plus ruxolitinib. The median duration of follow-up was 6.1 (range, 1.9 to 18.6) months. Twenty-eight (88%) patients received a navitoclax dose of 200 mg, and 4 (13%) received 100 mg OD. The median age was 69 years (range, 44 to 83), median spleen volume was 1889.08 cm3 (range, 645.6 to 7339.6), and 38% had at least 1 prior line of therapy. The median navitoclax and ruxolitinib exposures were 24.1 (range, 5.1 to 80.9) and 24.1 (5.1 to 80.9) weeks, respectively. Thirty-one (97%) patients experienced ≥1 AE, 25 (78%) had Grade ≥3 AEs, and 6 (19%) had serious AEs. The most frequent Grade ≥3 AEs were anemia (34%), thrombocytopenia (31%), and neutropenia (19%). Three (9%) and 2 (6%) patients experienced an AE that led to navitoclax and ruxolitinib discontinuation, respectively, and 2 (6%; 1 progressive disease, 1 cardiac disorder unrelated to navitoclax) AEs led to death ≤30 days after the last dose of navitoclax. SVR35 was achieved by 52% of evaluable patients at week 24 (SVR35 in DIPSS Intermediate-2, 50%; High-risk, 33%) and by 76% at any time on the study (Table). The median time to first SVR35 was 12.1 (range, 11 to 47) weeks. Image:Summary/Conclusion: The combination of navitoclax and ruxolitinib was well tolerated and demonstrated early and robust reductions in spleen volume, anemia, and BM fibrosis in patients without prior JAK-2 inhibitor exposure. SVR35, anemia, and bone marrow fibrosis improved over time.

Navitoclax plus ruxolitinib in JAK inhibitor-naive patients with myelofibrosis: Preliminary safety and efficacy in a multicenter, open-label phase 2 study.

7015 Background: Ruxolitinib (RUX), a Janus kinase (JAK) 1/2 inhibitor, is the current standard of care for patients (pts) with myelofibrosis (MF) that improves splenomegaly and disease symptoms with limited impact on disease biology. Many pts lose response over time, highlighting an unmet need for novel therapies. Navitoclax (NAV) is an oral, small-molecule inhibitor of BCL-XL and BCL-2 that has a synergistic effect when used in combination with JAK inhibitors to enhance apoptosis. This ongoing, open-label, multicenter, phase 2 trial (NCT03222609) is evaluating the efficacy and safety of NAV with/without RUX in pts with MF. Here, we report results from JAK inhibitor-naïve pts treated with NAV+RUX. Methods: Enrolled pts had primary or secondary MF with splenomegaly (DIPSS ≥INT-1) and did not receive prior JAK-2 therapy or bromodomain and extraterminal motif (BET) inhibitors. Pts initiated NAV at 100 mg QD or 200 mg QD if baseline (BL) platelet count was ≤150 × 109/L or >150 × 109/L, respectively. RUX was given BID with starting dose based on BL platelet count per local label. The primary endpoint was spleen volume reduction of ≥35% (SVR35) from BL at wk 24. Key secondary endpoints were ≥50% reduction in total symptom score (TSS50), bone marrow (BM) fibrosis reduction, and anemia response. Adverse events (AEs) were monitored throughout the study. Results: As of Oct 04, 2021, 32 pts received NAV+RUX. Median duration of f/u was 6.1 (range, 1.9 ─ 18.6) mos. 28 (88%) pts received NAV 200 mg and 4 (13%) received 100 mg OD. Median age was 69 (44 ─ 83) yrs, and median spleen volume was 1889.08 cm3 (645.6 ─ 7339.6). Median NAV and RUX exposures were 24.1 (5.1 ─ 80.9) and 20.1 (0.1 ─ 80.1) wks, respectively. 31 (97%) pts reported ≥1 AE (Grade ≥3 AEs, 25 [78%]; serious AEs, 6 [19%]). Most common Grade ≥3 AEs were anemia (34%), thrombocytopenia (31%), and neutropenia (19%). 3 (9%) and 2 (6%) pts reported an AE leading to NAV and RUX discontinuation, respectively, and 2 (6%; 1 PD, 1 cardiac disorder unrelated to NAV) AEs led to death ≤30 days after last NAV dose. SVR35 was achieved by 52% of evaluable pts at wk 24 (SVR35 in INT-2, 50%; HR, 33%) and by 76% at any time on treatment (Table). Median time to first SVR35 was 12.1 (11 ─ 47) wks. Conclusions: The combination of NAV+RUX was well tolerated and demonstrated early and robust reductions in spleen volume, anemia, and BM fibrosis in pts without prior JAK-2 inhibitor exposure. SVR35, TSS50, and BM fibrosis improved over time. Clinical trial information: NCT03222609. [Table: see text]

Chromatin Rewiring by Mismatch Repair Protein MSH2 Alters Cell Adhesion Pathways and Sensitivity to BET Inhibition in Gastric Cancer.

DNA repair protein MSH2 binds and regulates cell adhesion genes by enabling enhancer-promoter interactions, and loss of MSH2 causes deficient cell adhesion and bromodomain and extraterminal motif inhibitor synthetic lethality in gastric cancer.

Bromodomain Inhibitor JQ1 Provides Novel Insights and Perspectives in Rhabdomyosarcoma Treatment.

Rhabdomyosarcoma (RMS) is the most common type of pediatric soft tissue sarcoma. It is classified into two main subtypes: embryonal (eRMS) and alveolar (aRMS). MYC family proteins are frequently highly expressed in RMS tumors, with the highest levels correlated with poor prognosis. A pharmacological approach to inhibit MYC in cancer cells is represented by Bromodomain and Extra-Terminal motif (BET) protein inhibitors. In this paper, we evaluated the effects of BET inhibitor (+)-JQ1 (JQ1) on the viability of aRMS and eRMS cells. Interestingly, we found that the drug sensitivity of RMS cell lines to JQ1 was directly proportional to the expression of MYC. JQ1 induces G1 arrest in cells with the highest steady-state levels of MYC, whereas apoptosis is associated with MYC downregulation. These findings suggest BET inhibition as an effective strategy for the treatment of RMS alone or in combination with other drugs.

Loss of H3K27 Trimethylation Promotes Radiotherapy Resistance in Medulloblastoma and Induces an Actionable Vulnerability to BET Inhibition.

This study demonstrates a novel epigenetic mechanism of radiation resistance in medulloblastoma and identifies a therapeutic approach to improve outcomes in these patients.