Birabresib dihydrate

Despite advances in multidisciplinary care, the risk of late recurrence in Estrogen Receptor-positive (ER+) breast cancer (BC) patients following standard-of-care treatment ranges from 20-52% at 20 years. While immune checkpoint inhibitors (CPI), have shown enormous potential in inducing long term remission in some cases, patients with ER+ BC show poor response to these agents. The objective of this study is to develop effective radiation therapy (RT)-based combination regimens that induce anti-tumor immunity against immunologically cold ER+ breast tumors.

We have previously shown that pharmacological inhibition of bromodomain and extraterminal domain (BET) family of epigenetic regulator proteins overcomes both endocrine therapy resistance and radiation resistance in pre-clinical models of ER+ BC by reversing pathological reprogramming of transcriptional and DNA repair pathways, respectively. In this study, we explore the potential of RT+BET inhibition combination to elicit anti-tumor immunity against ER+ BC.

A unique ER+ BC model, MXT+, was used to generate tumors in female, BDF1 mice. PD-L1 expression following RT or RT+OTX015 (a BET inhibitor) was assessed by Western blot analysis. Comet assay was used to assess the degree of unrepaired DNA damage following treatment with RT alone, OTX015 alone and RT+OTX015. Multiplex IHC was used to assess changes in tumor microenvironment after various treatments. Tumor growth characteristics were established following treatment with vehicle, RT alone (8 Gy x 3), OTX015 alone (100 mg/kg x 3 days) and RT+OTX015.

Using murine ER+ BC model, MXT+, we show that RT is a potent inducer of PD-L1 expression. Combination of RT+OTX015 completely ablated RT-mediated PD-L1 induction at the transcription level. OTX015 also accentuated RT-induced DNA damage through inhibition of NHEJ pathway, resulting in increased micronuclei formation (which activate cGAS/STING pathway, a critical bridge between innate and adaptive immunity). Combination of OTX015 with RT also caused a statistically significant (on unpaired t test) increase tumor infiltrating lymphocytes. In in vivo therapeutic studies, combination of OTX015 with RT (8 Gy x 3) resulted in extraordinary synergy and caused complete inhibition of long-term tumor growth. Treatment with RT alone (8 Gy x 3) or OTX015 alone (on 3 consecutive days) only caused modest inhibition of tumor growth. ANOVA with Dunnett's test was used to adjust for multiple comparisons and showed statistically significant difference in tumor growth between OTX015+RT arm and OTX alone as well as OTX015+RT arm and RT alone.

Patients with ER+ BC face high risk of late recurrence after definitive treatment and show poor response to immune CPIs. We show that combination of BET inhibition with RT induces anti-tumor immunity and completely prevents tumor growth. Clinical validation of this regimen is warranted to prevent late recurrences in ER+ BC patients.

Kaposi sarcoma-associated herpesvirus (KSHV) inflammatory cytokine syndrome (KICS) is a newly described chronic inflammatory disease condition caused by KSHV infection and is characterized by high KSHV viral load and sustained elevations of serum KSHV-encoded IL-6 (vIL-6) and human IL-6 (hIL-6). KICS has significant immortality and possesses greater risks of having other complications, which include malignancies. Although prolonged inflammatory vIL-6 exposure by persistent KSHV infection is expected to have key roles in subsequent disease development, the biological effects of prolonged vIL-6 exposure remain elusive. Using thiol-Linked Alkylation for the Metabolic Sequencing and Cleavage Under Target & Release Using Nuclease analysis, we studied the effect of prolonged vIL-6 exposure in chromatin landscape and resulting cytokine production. The studies showed that prolonged vIL-6 exposure increased Bromodomain containing 4 (BRD4) and histone H3 lysine 27 acetylation co-occupancies on chromatin, and the recruitment sites were frequently co-localized with poised RNAPII with associated enzymes. Increased BRD4 recruitment on promoters was associated with increased and prolonged NF-KB p65 binding after the lipopolysaccharide stimulation. The p65 binding resulted in quicker and sustained transcription bursts from the promoters; this mechanism increased total amounts of hIL-6 and IL-10 in tissue culture. Pretreatment with the BRD4 inhibitor, OTX015, eliminated the enhanced inflammatory cytokine production. These findings suggest that persistent vIL-6 exposure may establish a chromatin landscape favorable for the reactivation of inflammatory responses in monocytes. This epigenetic memory may explain the greater risk of chronic inflammatory disease development in KSHV-infected individuals.

Combined and continuous cytokine stimulation triggers transcription reprogramming and is often used for specific tissue development. Continuous vIL-6 exposure occurs in KSHV-infected patients and leads to inflammatory cytokine storm with high mortality. However, possible epigenetic reprogramming by the vIL-6 and its association with pathogenesis remain unclear. Here we demonstrate the establishment of a new chromatin landscape mediated by BRD4 through prolonged vIL-6 exposure which contributes to more robust and rapid transcription and increased cytokines production. Inhibition of BRD4 suppressed this inflammatory response. Our results indicate that targeting the epigenetic effect of viral cytokines may lead to novel therapies for KSHV-induced inflammatory cytokine storms.