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Effective therapeutics are urgently needed to counter infection and improve outcomes for patients suffering from COVID-19 and to combat this pandemic. Manipulation of epigenetic machinery to influence viral infectivity of host cells is a relatively unexplored area. The bromodomain and extraterminal (BET) family of epigenetic readers have been reported to modulate SARS-CoV-2 infection. Herein, we demonstrate apabetalone, the most clinical advanced BET inhibitor, downregulates expression of cell surface receptors involved in SARS-CoV-2 entry, including angiotensin-converting enzyme 2 (ACE2) and dipeptidyl-peptidase 4 (DPP4 or CD26) in SARS-CoV-2 permissive cells. Moreover, we show that apabetalone inhibits SARS-CoV-2 infection in vitro to levels comparable to those of antiviral agents. Taken together, our study supports further evaluation of apabetalone to treat COVID-19, either alone or in combination with emerging therapeutics.

B-cell chronic lymphocytic leukemia (CLL) results from intrinsic genetic defects and complex microenvironment stimuli that fuel CLL cell growth through an array of survival signaling pathways. Novel small-molecule agents targeting the B-cell receptor pathway and anti-apoptotic proteins alone or in combination have revolutionized the management of CLL, yet combination therapy carries significant toxicity and CLL remains incurable due to residual disease and relapse. Single-molecule inhibitors that can target multiple disease-driving factors are thus an attractive approach to combat both drug resistance and combination-therapy-related toxicities. We demonstrate that SRX3305, a novel small-molecule BTK/PI3K/BRD4 inhibitor that targets three distinctive facets of CLL biology, attenuates CLL cell proliferation and promotes apoptosis in a dose-dependent fashion. SRX3305 also inhibits the activation-induced proliferation of primary CLL cells in vitro and effectively blocks microenvironment-mediated survival signals, including stromal cell contact. Furthermore, SRX3305 blocks CLL cell migration toward CXCL-12 and CXCL-13, which are major chemokines involved in CLL cell homing and retention in microenvironment niches. Importantly, SRX3305 maintains its anti-tumor effects in ibrutinib-resistant CLL cells. Collectively, this study establishes the preclinical efficacy of SRX3305 in CLL, providing significant rationale for its development as a therapeutic agent for CLL and related disorders.

There was a trend toward a slight improvement in neutrophil engraftment post-ABMT with the PIXY321 administered by an SC route compared with GM-CSF administered by an IV route. However, no differences could be identified between the two agents with respect to the time to platelet transfusion independence. Patient, regimen, and graft characteristics were most predictive of the engraftment tempo.

The COVID-19 pandemic caused by SARS-CoV-2 infection has led to socioeconomic shutdowns and the loss of over 5 million lives worldwide. There is a need for the identification of therapeutic targets to treat COVID-19. SARS-CoV-2 spike is a target of interest for the development of therapeutic targets.We developed a robust SARS-CoV-2 S spike expression and purification protocol from insect cells and studied four recombinant SARS-CoV-2 spike protein constructs based on the original SARS-CoV-2 sequence using a baculovirus expression system: a spike protein receptor-binding domain that includes the SD1 domain (RBD) coupled to a fluorescent tag (S-RBD-eGFP), spike ectodomain coupled to a fluorescent tag (S-Ecto-eGFP), spike ectodomain with six proline mutations and a foldon domain (S-Ecto-HexaPro(+F)), and spike ectodomain with six proline mutations without the foldon domain (S-Ecto-HexaPro(-F)). We tested the yield of purified protein expressed from the insect cell lines Spodoptera frugiperda (Sf9) and Trichoplusia ni (Tni) and compared it to previous research using mammalian cell lines to determine changes in protein yield. We demonstrated quick and inexpensive production of functional glycosylated spike protein of high purity capable of recognizing and binding to the angiotensin converting enzyme 2 (ACE2) receptor. To further confirm functionality, we demonstrate binding of eGFP fused construct of the spike ectodomain (S-Ecto-eGFP) to surface ACE2 receptors on lung epithelial cells by flow cytometry analysis and show that it can be decreased by means of receptor manipulation (blockade or downregulation).

Mantle cell lymphoma (MCL) is a subtype of non-Hodgkin’s lymphoma characterized by poor prognosis. The complexity of MCL pathogenesis arises from aberrant activities of diverse signaling pathways, including BTK, PI3K–AKT–mTOR and MYC-BRD4. Here, we report that MCL-related signaling pathways can be altered by a single small molecule inhibitor, SRX3305. Binding and kinase activities along with resonance changes in NMR experiments reveal that SRX3305 targets both bromodomains of BRD4 and is highly potent in inhibition of the PI3K isoforms α, γ and δ, as well as BTK and the drug-resistant BTK mutant. Preclinical investigations herein reveal that SRX3305 perturbs the cell cycle, promotes apoptosis in MCL cell lines and shows dose dependent anti-proliferative activity in both MCL and drug-resistant MCL cells. Our findings underscore the effectiveness of novel multi-action small molecule inhibitors for potential treatment of MCL.

Background: The chronic lymphocytic leukemia (CLL) tumor microenvironment (TME) is laden with hyporesponsive T-cells that permit disease persistence. Yet, redundant TME immunosuppressive mechanisms and epigenetic maintenance of T-cell exhaustion limit the efficacy of T-cell targeted therapies in CLL. Bromodomain and extra-terminal (BET) proteins regulate key pathways contributing to CLL pathogenesis and TME interactions, including T-cell function and differentiation. We hypothesize that blocking BET protein function can reverse T-cell exhaustion to yield durable tumor elimination in CLL. Methods: WT C57BL/6 mice were engrafted with Eμ-TCL1 spleen-derived lymphocytes, then treated daily with the novel pan-BET inhibitor, OPN-51107 (OPN5; 20mg/kg PO) for up to 4 weeks. Splenic gene expression was evaluated with the NanoString PanCancer iO360 panel. T-cell differentiation status, immune inhibitory receptor (IR) expression, proliferation (72 h ex vivo α-CD3/α-CD28 stimulation), and cytokine production (6 h ex vivo PMA/ionomycin stimulation) was measured via flow cytometry. CLL patient and healthy donor PBMCs were used for validation studies and to assess T-cell transcription factor (TF) expression via flow cytometry. Evaluation of BRD4 occupancy at select T-cell TFs via ChIP qPCR is ongoing. Results: OPN5 significantly increased cytotoxic cell signatures and reduced exhaustion-associated cell signatures in leukemic mice through inhibition of T-cell exhaustion signaling, as well as activation of Th1, natural killer cell, and IL-7 signaling pathways. Correspondingly, T-cells from OPN5-treated mice demonstrated greater ex vivo proliferative capacity and effector response to stimuli. A greater proportion of CD8+ T-cells from OPN5-treated mice were classified as naïve, and OPN5 significantly reduced KLRG1 expression on antigen-experienced CD8+ T-cells. Importantly, OPN5 curtailed IR co-expression (PD-1, PD-L1, VISTA, CD244, CD160, and LAG3) on splenic T-cells. These findings were confirmed with primary CLL cells ex vivo. OPN5 also impaired expression of terminal differentiation-associated TFs in CLL patient-derived T-cells, enriching for a TCF1+ progenitor T-cell population. While BTK inhibitors are known to similarly improve T-cell function in CLL, ibrutinib treatment was inadequate to revert CLL T-cell terminal differentiation. Future ATAC-sequencing analysis will inform how BET inhibition alleviates exhaustion-associated chromatin organization in CLL T-cells. Conclusion: BET inhibition dismantles immunosuppressive mechanisms in the CLL TME, alleviating CLL-induced T-cell dysfunction and terminal differentiation. These findings suggest that BET inhibition may be a useful component of combination strategies for the treatment of CLL to yield lasting anti-cancer immune memory and prevent relapsed/refractory disease. Citation Format: Audrey L. Smith, Alexandria P. Eiken, Sydney A. Skupa, Christopher R. D'Angelo, Avyakta Kallam, Matthew A. Lunning, Gregory Bociek, Julie M. Vose, Ben Powell, Gideon Bollag, Dalia El-Gamal. BET inhibition alleviates T-cell dysfunction in chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6396.

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