Tríona Ní Chonghaile

Histone deacetylase 6 (HDAC6) is a cytoplasmic enzyme that deacetylates non-histone substrates such as α-tubulin and cortactin. HDAC6 contains two catalytic domains, each containing a catalytic zinc ion, and a zinc-finger ubiquitin-binding domain. We have discovered BAS-2, a selective HDAC6 inhibitor with an isothiouronium core and no obvious zinc-binding group. To define its mechanism, we combine X-ray crystallography, structure-activity-relationships, molecular modeling and mutagenesis. BAS-2 potently inhibits human HDAC6 but it does not inhibit zebrafish HDAC6. Computational modeling highlighted Asp567 in human HDAC6 as critical for BAS-2 recognition and mutational analyses confirmed this. The corresponding zebrafish residue is Asn530 and the crystal structure of the N530D variant zHDAC6 revealed binding of a BAS-2–derived mercaptoacetamide that engages the catalytic zinc via strong thiolate–zinc coordination. Leveraging the orientation of BAS-2 binding, we designed a BAS-2–based proteolysis targeting chimera that induced proteasome-dependent HDAC6 degradation in cells, verified by global proteomics. Collectively, these insights clarify species selectivity and demonstrate that BAS-2 acts as a selective, mechanism-based inhibitor of human HDAC6. These discoveries will aid the development of the next generation of selective HDAC6 inhibitors and degraders.

Breast cancer is the most frequently diagnosed malignancy and a leading cause of cancer-related mortality among women worldwide. Triple-negative breast cancer (TNBC) poses a major clinical challenge due to its aggressive nature, limited therapeutic options, and high propensity for drug resistance. Dysregulation of the PI3K/AKT/mTOR and HDAC signalling pathways has been implicated in TNBC progression and therapeutic resistance, highlighting their potential as combinatorial targets. In this study, we report the design, synthesis, and biological evaluation of a novel series of triazine-based multitarget inhibitors aimed at the dual inhibition of PI3K and HDAC. Among the synthesized compounds, 5b and 5f demonstrated the most promising profiles, exhibiting low nanomolar IC50 values against HDAC6 (2.33 and 6.02 nM) and PI3Kα (17.5 and 236 nM), respectively. Both compounds reduced cell viability in breast cancer cell lines, with IC₅₀ values below 5 µM in MDA-MB-231 cells. Western blot analysis confirmed inhibition of HDAC and PI3K signalling in treated cells. Molecular docking and dynamics simulations further revealed stable binding modes and favourable interactions within the active sites of both targets. Overall, 5b and 5f represent promising lead candidates for further optimization toward the development of selective dual HDAC6/PI3K inhibitors with potential application in TNBC therapy, by evaluation in TNBC-relevant models.

Fumarate hydratase (FH), a key node of mitochondrial metabolism, is also a tumour suppressor. Despite its prominent roles in tumourigenesis and inflammation, its regulation remains poorly understood. Herein, we show that histone deacetylase 6 (HDAC6) regulates FH activity. In triple-negative breast cancer cells, HDAC6 inhibition or knockdown results in alterations to mitochondrial cristae structure, as detected by live-cell super-resolution STED nanoscopy and electron microscopy, along with the release of mitochondrial DNA. Mass-spectrometry immunoprecipitation reveals multiple mitochondrial HDAC6-interactors, with FH emerging as a top hit. Super-resolution 3D-STORM shows HDAC6 interactions with FH in mitochondrial networks, which increases after perturbation of HDAC6 activity with BAS-2. Treatment with BAS-2 leads to fumarate accumulation by ¹³C glucose labelling, along with downstream succination of proteins and cell death. Together, these results identify HDAC6 inhibition as a regulator of endogenous FH activity in tumour cells, and highlight it as a promising candidate for indirectly targeting tumour metabolism.

Multiple myeloma (MM) is an incurable plasma cell malignancy that, despite an unprecedented increase in overall survival, lacks truly risk-adapted or targeted treatments. A proportion of patients with MM depend on BCL-2 for survival, and, recently, the BCL-2 antagonist venetoclax has shown clinical efficacy and safety in t(11;14) and BCL-2 overexpressing MM. However, only a small proportion of MM patients rely on BCL-2 (approx. 20%), and there is a need to broaden the patient population outside of t(11;14) that can be treated with venetoclax. Therefore, we took an unbiased screening approach and screened epigenetic modifiers to enhance venetoclax sensitivity in 2 non-BCL-2 dependent MM cell lines. The demethylase inhibitor 5-azacytidine was one of the lead hits from the screen, and the enhanced cell killing of the combination was confirmed in additional MM cell lines. Using dynamic BH3 profiling and immunoprecipitations, we identified the potential mechanism of synergy is due to increased NOXA expression, through the integrated stress response. Knockdown of PMAIP1 or PKR partially rescues cell death of the venetoclax and 5-azacytidine combination treatment. The addition of a steroid to the combination treatment did not enhance the cell death, and, interestingly, we found enhanced death of the immune cells with steroid addition, suggesting that a steroid-sparing regimen may be more beneficial in MM. Lastly, we show for the first time in primary MM patient samples that 5-azacytidine enhances the response to venetoclax ex vivo across diverse anti-apoptotic dependencies (BCL-2 or MCL-1) and diverse cytogenetic backgrounds. Overall, our data identify 5-azacytidine and venetoclax as an effective treatment combination, which could be a tolerable steroid-sparing regimen, particularly for elderly MM patients.

The targeted degradation of proteins bound by metals represents a promising approach to treat diseases. We report the development of the first metallo-PROTAC, specifically a Pt-PROTAC, that can effectively degrade select Pt(II)-binding proteins. The reported Pt-PROTAC prototype successfully degraded thioredoxin-1 and thioredoxin reductase-1 though not glutathione-S-transferase in JJN3 and MM1.S multiple myeloma cancer cell lines. Deactivated Pt-PROTAC does not degrade thioredoxin-1 and thioredoxin reductase-1. Furthermore pretreatment of cells with the proteasome inhibitor bortezomib prevents Pt-PROTAC target degradation thereby implicating the ubiquitin proteasome system with its mode of degradation. Metallo-PROTACS will have important applications in the identification of metal binding proteins and as chemotherapeutic agents.

Venetoclax is a B cell lymphoma 2 (BCL-2)–selective antagonist used to treat chronic lymphocytic leukemia (CLL) and acute myelogenous leukemia (AML). Although this has been a promising therapeutic option for these patients, many of these patients develop resistance and relapsed disease. Here, we summarize the emerging mechanisms of resistance to venetoclax treatment, discuss the promising combination strategies, and highlight the combinations that are currently in clinical trials. Efforts to understand mechanisms of resistance are critical to advance the development of new targeted therapeutic strategies and further our understanding of the biological functions of BCL-2 in tumor cells.

: Due to developments in modern chemistry, previously uundruggable substrates are now targetable thanks to selective degradation using the ubiquitin-proteasomal degradation system. PROteolysis TArgeting Chimeras (PROTACs) are heterobifunctional molecules designed specifically to degrade target proteins. They are of significant interest to industry and academia as they are highly specific and can target previously undruggable target proteins from transcription factors to enzymes. More than 15 degraders are expected to be evaluated in clinical trials by the end of 2021. Herein, we describe recent advances in the design and development of PROTAC-mediated degradation of histone deacetylases (HDACs). PROTAC-mediated degradation of HDACs can offer some significant advantages over direct inhibition, such as the use of substoichiometric doses and the potential to disrupt enzyme-independent HDAC function. We discuss the potential implication of the degradation of HDACs in comparison with HDAC knockout studies. Along with the selection of HDAC inhibitors and E3 ligase ligands for the design of PROTACs. The potential utility of HDAC PROTACs in various disease pathologies from cancer to inflammation to neurodegeneration is driving the interest in this field.

B-cell lymphoma 2 (BCL-2) has recently emerged as a therapeutic target for early T-cell progenitor acute lymphoblastic leukemia (ETP-ALL), a high-risk subtype of human T-cell ALL. The major clinical challenge with targeted therapeutics, such as the BCL-2 inhibitor ABT-199, is the development of acquired resistance. We assessed the in vivo response of luciferase-positive LOUCY cells to ABT-199 monotherapy and observed specific residual disease in the splenic microenvironment. Of note, these results were confirmed by using a primary ETP-ALL patient-derived xenograft. Splenomegaly has previously been associated with poor prognosis in diverse types of leukemia. However, the exact mechanism by which the splenic microenvironment alters responses to specific targeted therapies remains largely unexplored. We show that residual LOUCY cells isolated from the spleen microenvironment displayed reduced BCL-2 dependence, which was accompanied by decreased BCL-2 expression levels. Notably, this phenotype of reduced BCL-2 dependence could be recapitulated by using human splenic fibroblast coculture experiments and was confirmed in an in vitro chronic ABT-199 resistance model of LOUCY. Finally, single-cell RNA-sequencing was used to show that ABT-199 triggers transcriptional changes in T-cell differentiation genes in leukemic cells obtained from the spleen microenvironment. Of note, increased expression of CD1a and sCD3 was also observed in ABT199-resistant LOUCY clones, further reinforcing the idea that a more differentiated leukemic population might display decreased sensitivity toward BCL-2 inhibition. Overall, our data reveal the spleen as a site of residual disease for ABT-199 treatment in ETP-ALL and provide evidence for plasticity in T-cell differentiation as a mechanism of therapy resistance.

Discovery of a new HDAC6 inhibitor reveals role for HDAC6 in the regulation of glycolytic metabolism.

Approximately 70% of breast cancers express estrogen receptor α (ERα) and depend on this key transcriptional regulator for proliferation and differentiation. While patients with this disease can be treated with targeted antiendocrine agents, drug resistance remains a significant issue, with almost half of patients ultimately relapsing. Elucidating the mechanisms that control ERα function may further our understanding of breast carcinogenesis and reveal new therapeutic opportunities. Here, we investigated the role of deubiquitinases (DUB) in regulating ERα in breast cancer. An RNAi loss-of-function screen in breast cancer cells targeting all DUBs identified USP11 as a regulator of ERα transcriptional activity, which was further validated by assessment of direct transcriptional targets of ERα. USP11 expression was induced by estradiol, an effect that was blocked by tamoxifen and not observed in ERα-negative cells. Mass spectrometry revealed a significant change to the proteome and ubiquitinome in USP11-knockdown (KD) cells in the presence of estradiol. RNA sequencing in LCC1 USP11-KD cells revealed significant suppression of cell-cycle–associated and ERα target genes, phenotypes that were not observed in LCC9 USP11-KD, antiendocrine-resistant cells. In a breast cancer patient cohort coupled with in silico analysis of publicly available cohorts, high expression of USP11 was significantly associated with poor survival in ERα-positive (ERα+) patients. Overall, this study highlights a novel role for USP11 in the regulation of ERα activity, where USP11 may represent a prognostic marker in ERα+ breast cancer. Significance: A newly identified role for USP11 in ERα transcriptional activity represents a novel mechanism of ERα regulation and a pathway to be exploited for the management of ER-positive breast cancer.