From PROTAC to inhibitor: Structure-guided discovery of potent and orally bioavailable BET inhibitors.

An X-ray structure of a CLICK chemistry-based BET PROTAC bound to BRD2(BD2) inspired synthesis of JQ1 derived heterocyclic amides. This effort led to the discovery of potent BET inhibitors displaying overall improved profiles when compared to JQ1 and birabresib. A thiadiazole derived 1q (SJ1461) displayed excellent BRD4 and BRD2 affinity and high potency in the panel of acute leukaemia and medulloblastoma cell lines. A structure of 1q co-crystalised with BRD4-BD1 revealed polar interactions with the AZ/BC loops, in particular with Asn140 and Tyr139, rationalising the observed affinity improvements. In addition, exploration of pharmacokinetic properties of this class of compounds suggest that the heterocyclic amide moiety improves drug-like features. Our study led to the discovery of potent and orally bioavailable BET inhibitor 1q (SJ1461) as a promising candidate for further development.

Combination of Ribociclib with BET-Bromodomain and PI3K/mTOR Inhibitors for Medulloblastoma Treatment In Vitro and In Vivo.

Despite improvement in the treatment of medulloblastoma over the last years, numerous patients with MYC- and MYCN-driven tumors still fail current therapies. Medulloblastomas have an intact retinoblastoma protein RB, suggesting that CDK4/6 inhibition might represent a therapeutic strategy for which drug combination remains understudied. We conducted high-throughput drug combination screens in a Group3 (G3) medulloblastoma line using the CDK4/6 inhibitor (CDK4/6i) ribociclib at IC20, referred to as an anchor, and 87 oncology drugs approved by FDA or in clinical trials. Bromodomain and extra terminal (BET) and PI3K/mTOR inhibitors potentiated ribociclib inhibition of proliferation in an established cell line and freshly dissociated tumor cells from intracranial xenografts of G3 and Sonic hedgehog (SHH) medulloblastomas in vitro. A reverse combination screen using the BET inhibitor JQ1 as anchor, revealed CDK4/6i as the most potentiating drugs. In vivo, ribociclib showed single-agent activity in medulloblastoma models whereas JQ1 failed to show efficacy due to high clearance and insufficient free brain concentration. Despite in vitro synergy, combination of ribociclib with the PI3K/mTOR inhibitor paxalisib did not significantly improve the survival of G3 and SHH medulloblastoma-bearing mice compared with ribociclib alone. Molecular analysis of ribociclib and paxalisib-treated tumors revealed that E2F targets and PI3K/AKT/MTORC1 signaling genes were depleted, as expected. Importantly, in one untreated G3MB model HD-MB03, the PI3K/AKT/MTORC1 gene set was enriched in vitro compared with in vivo suggesting that the pathway displayed increased activity in vitro. Our data illustrate the difficulty in translating in vitro findings in vivo. See related article in Mol Cancer Ther (2022) 21(8):1306-1317.

Combination of Ribociclib and Gemcitabine for the Treatment of Medulloblastoma.

Group3 (G3) medulloblastoma (MB) is one of the deadliest forms of the disease for which novel treatment is desperately needed. Here we evaluate ribociclib, a highly selective CDK4/6 inhibitor, with gemcitabine in mouse and human G3MBs. Ribociclib central nervous system (CNS) penetration was assessed by in vivo microdialysis and by IHC and gene expression studies and found to be CNS-penetrant. Tumors from mice treated with short term oral ribociclib displayed inhibited RB phosphorylation, downregulated E2F target genes, and decreased proliferation. Survival studies to determine the efficacy of ribociclib and gemcitabine combination were performed on mice intracranially implanted with luciferase-labeled mouse and human G3MBs. Treatment of mice with the combination of ribociclib and gemcitabine was well tolerated, slowed tumor progression and metastatic spread, and increased survival. Expression-based gene activity and cell state analysis investigated the effects of the combination after short- and long-term treatments. Molecular analysis of treated versus untreated tumors showed a significant decrease in the activity and expression of genes involved in cell-cycle progression and DNA damage response, and an increase in the activity and expression of genes implicated in neuronal identity and neuronal differentiation. Our findings in both mouse and human patient-derived orthotopic xenograft models suggest that ribociclib and gemcitabine combination therapy warrants further investigation as a treatment strategy for children with G3MB.

Phenyl-Glutarimides: Alternative Cereblon Binders for the Design of PROTACs.

Targeting cereblon (CRBN) is currently one of the most frequently reported proteolysis-targeting chimera (PROTAC) approaches, owing to favorable drug-like properties of CRBN ligands, immunomodulatory imide drugs (IMiDs). However, IMiDs are known to be inherently unstable, readily undergoing hydrolysis in body fluids. Here we show that IMiDs and IMiD-based PROTACs rapidly hydrolyze in commonly utilized cell media, which significantly affects their cell efficacy. We designed novel CRBN binders, phenyl glutarimide (PG) analogues, and showed that they retained affinity for CRBN with high ligand efficiency (LE >0.48) and displayed improved chemical stability. Our efforts led to the discovery of PG PROTAC 4 c (SJ995973), a uniquely potent degrader of bromodomain and extra-terminal (BET) proteins that inhibited the viability of human acute myeloid leukemia MV4-11 cells at low picomolar concentrations (IC50 =3 pM; BRD4 DC50 =0.87 nM). These findings strongly support the utility of PG derivatives in the design of CRBN-directed PROTACs.

Patient-derived models recapitulate heterogeneity of molecular signatures and drug response in pediatric high-grade glioma.

Pediatric high-grade glioma (pHGG) is a major contributor to cancer-related death in children. In vitro and in vivo disease models reflecting the intimate connection between developmental context and pathogenesis of pHGG are essential to advance understanding and identify therapeutic vulnerabilities. Here we report establishment of 21 patient-derived pHGG orthotopic xenograft (PDOX) models and eight matched cell lines from diverse groups of pHGG. These models recapitulate histopathology, DNA methylation signatures, mutations and gene expression patterns of the patient tumors from which they were derived, and include rare subgroups not well-represented by existing models. We deploy 16 new and existing cell lines for high-throughput screening (HTS). In vitro HTS results predict variable in vivo response to PI3K/mTOR and MEK pathway inhibitors. These unique new models and an online interactive data portal for exploration of associated detailed molecular characterization and HTS chemical sensitivity data provide a rich resource for pediatric brain tumor research.

Identification of Potent, Selective, and Orally Bioavailable Small-Molecule GSPT1/2 Degraders from a Focused Library of Cereblon Modulators.

Whereas the PROTAC approach to target protein degradation greatly benefits from rational design, the discovery of small-molecule degraders relies mostly on phenotypic screening and retrospective target identification efforts. Here, we describe the design, synthesis, and screening of a large diverse library of thalidomide analogues against a panel of patient-derived leukemia and medulloblastoma cell lines. These efforts led to the discovery of potent and novel GSPT1/2 degraders displaying selectivity over classical IMiD neosubstrates, such as IKZF1/3, and high oral bioavailability in mice. Taken together, this study offers compound 6 (SJ6986) as a valuable chemical probe for studying the role of GSPT1/2 in vitro and in vivo, and it supports the utility of a diverse library of CRBN binders in the pursuit of targeting undruggable oncoproteins.

Bromodomain-Selective BET Inhibitors Are Potent Antitumor Agents against MYC-Driven Pediatric Cancer.

Inhibition of members of the bromodomain and extraterminal (BET) family of proteins has proven a valid strategy for cancer chemotherapy. All BET identified to date contain two bromodomains (BD; BD1 and BD2) that are necessary for recognition of acetylated lysine residues in the N-terminal regions of histones. Chemical matter that targets BET (BETi) also interact via these domains. Molecular and cellular data indicate that BD1 and BD2 have different biological roles depending upon their cellular context, with BD2 particularly associated with cancer. We have therefore pursued the development of BD2-selective molecules both as chemical probes and as potential leads for drug development. Here we report the structure-based generation of a novel series of tetrahydroquinoline analogs that exhibit >50-fold selectivity for BD2 versus BD1. This selective targeting resulted in engagement with BD-containing proteins in cells, resulting in modulation of MYC proteins and downstream targets. These compounds were potent cytotoxins toward numerous pediatric cancer cell lines and were minimally toxic to nontumorigenic cells. In addition, unlike the pan BETi (+)-JQ1, these BD2-selective inhibitors demonstrated no rebound expression effects. Finally, we report a pharmacokinetic-optimized, metabolically stable derivative that induced growth delay in a neuroblastoma xenograft model with minimal toxicity. We conclude that BD2-selective agents are valid candidates for antitumor drug design for pediatric malignancies driven by the MYC oncogene. SIGNIFICANCE: This study presents bromodomain-selective BET inhibitors that act as antitumor agents and demonstrates that these molecules have in vivo activity towards neuroblastoma, with essentially no toxicity.

Regulation of senescence escape by the cdk4-EZH2-AP2M1 pathway in response to chemotherapy.

Senescence is a tumor suppressive mechanism that induces a permanent proliferative arrest in response to an oncogenic insult or to the genotoxic stress induced by chemotherapy. We have recently described that some cells can escape this arrest, either because senescence was incomplete or as a consequence of a phenotypic adaptation. Malignant cells which resisted senescence emerged as more transformed cells that resist anoikis and rely on survival pathways activated by Akt and Mcl-1. In this study, we further characterize senescence escape, investigating how emergent cells could reproliferate. During the initial step of chemotherapy-induced senescence (CIS), we found that cyclin D1 was upregulated and that cell emergence was prevented when its main partner cdk4 was inactivated. Results indicate that this kinase induced the upregulation of the EZH2 methylase, a component of the polycomb PRC2 complex. Downregulated during the early step of treatment, the methylase was reactivated in clones that escaped senescence. The inactivation of EZH2, either by siRNA or by specific inhibitors, led to a specific inhibition of cell emergence. We used quantitative proteomic analysis to identify new targets of the methylase involved in senescence escape. We identified proteins involved in receptor endocytosis and described new functions for the AP2M1 protein in the control of chemotherapy-mediated senescence. Our results indicate that AP2M1 is involved in the transmission of secreted signals produced by senescent cells, suggesting that this pathway might regulate specific receptors involved in the control of CIS escape. In light of these results, we therefore propose that the cdk4-EZH2-AP2M1 pathway plays an important role during chemotherapy resistance and senescence escape. Since targeted therapies are available against these proteins, we propose that they should be tested in the treatment of colorectal or breast cancers that become resistant to first-line genotoxic therapies.

[Contribution to tumor escape and chemotherapy response: A choice between senescence and apoptosis in heterogeneous tumors]. / Échappement tumoral lors de la chimiothérapie: un choix entre sénescence et apoptose dans des tumeurs hétérogènes.

Understanding adaptive signaling pathways in response to chemotherapy is one of the main challenges of cancer treatment. Activated in response to DNA damage, cell cycle and mitotic checkpoints activate the p53-p21 and p16-Rb pathways and induce apoptosis or senescence. Since senescent cells survive and produce a secretome that influences neighbouring cells, it is not particularly clear whether these responses are equivalent and if tumor cells escape these two suppressive pathways to the same extent. Predicting escape is also complicated by the fact that cancer cells adapt to treatments by activating the epithelial-mesenchymal transition and by producing clones with cancer-initiating cells features. Dedifferentiation pathways used in stressful conditions reconstitute dividing and sometimes more aggressive populations in response to chemotherapy. These observations illustrate the importance of tumor heterogeneity and the adaptation capacities of different intra-tumoral subclones. Depending on their oncogenic profile, on their localisation within the tumor and on their interaction with stromal cells, these subclones are expected to have different responses and adaptation capacities to chemotherapy. A complete eradication will certainly rely on combination therapies that can kill at the same time the bulk of the sensitive tumor but can also prevent plasticity and the generation of persistent clones.

Akt inhibition improves irinotecan treatment and prevents cell emergence by switching the senescence response to apoptosis.

Activated in response to chemotherapy, senescence is a tumor suppressive mechanism that induces a permanent loss of proliferation. However, in response to treatment, it is not really known how cells can escape senescence and how irreversible or incomplete this pathway is. We have recently described that cells that escape senescence are more transformed than non-treated parental cells, they resist anoikis and rely on Mcl-1. In this study, we further characterize this emergence in response to irinotecan, a first line treatment used in colorectal cancer. Our results indicate that Akt was activated as a feedback pathway during the early step of senescence. The inhibition of the kinase prevented cell emergence and improved treatment efficacy, both in vitro and in vivo. This improvement was correlated with senescence inhibition, p21waf1 downregulation and a concomitant activation of apoptosis due to Noxa upregulation and Mcl-1 inactivation. The inactivation of Noxa prevented apoptosis and increased the number of emergent cells. Using either RNA interference or p21waf1-deficient cells, we further confirmed that an intact p53-p21-senescence pathway favored cell emergence and that its downregulation improved treatment efficacy through apoptosis induction. Therefore, although senescence is an efficient suppressive mechanism, it also generates more aggressive cells as a consequence of apoptosis inhibition. We therefore propose that senescence-inducing therapies should be used sequentially with drugs favoring cell death such as Akt inhibitors. This should reduce cell emergence and tumor relapse through a combined induction of senescence and apoptosis.

Irinotecan treatment and senescence failure promote the emergence of more transformed and invasive cells that depend on anti-apoptotic Mcl-1.

Induction of senescence by chemotherapy was initially characterized as a suppressive response that prevents tumor cell proliferation. However, in response to treatment, it is not really known how cells can survive senescence and how irreversible this pathway is. In this study, we analyzed cell escape in response to irinotecan, a first line treatment used in colorectal cancer that induced senescence. We detected subpopulations of cells that adapted to chemotherapy and resumed proliferation. Survival led to the emergence of more transformed cells that induced tumor formation in mice and grew in low adhesion conditions. A significant amount of viable polyploid cells was also generated following irinotecan failure. Markers such as lgr5, CD44, CD133 and ALDH were downregulated in persistent clones, indicating that survival was not associated with an increase in cancer initiating cells. Importantly, malignant cells which resisted senescence relied on survival pathways induced by Mcl-1 signaling and to a lesser extent by Bcl-xL. Depletion of Mcl-1 increased irinotecan efficiency, induced the death of polyploid cells, prevented cell emergence and inhibited growth in low-adhesion conditions. We therefore propose that Mcl-1 targeting should be considered in the future to reduce senescence escape and to improve the treatment of irinotecan-refractory colorectal cancers.

STAT3 as a new autophagy regulator.

Signal transducers and activators of transcription 3 (STAT3) proteins are cytoplasmic transcription factors that translocate into the nucleus to induce transcription following growth factor or cytokine stimulation. Besides their normal functions, these proteins play an important role in cancer cells through the abnormal activation of cell cycle progression and the deregulation of survival and senescence pathways. New data obtained from the laboratory of Guido Kroemer identifies STAT3 as a new autophagy regulator. In the cytoplasm, in the absence of conventional phosphorylation on the tyrosine 705 residue, STAT3 interacts with the PKR kinase to inhibit eIF2A phosphorylation and so reduce autophagic pathways. This new and nonconventional function of STAT3 has an important role in normal cells but we suggest that it might also affect cancer cells and the response to chemotherapy treatment.

Distinct merkel cell polyomavirus molecular features in tumour and non tumour specimens from patients with merkel cell carcinoma.

Merkel Cell Polyomavirus (MCPyV) is associated with Merkel Cell carcinoma (MCC), a rare, aggressive skin cancer with neuroendocrine features. The causal role of MCPyV is highly suggested by monoclonal integration of its genome and expression of the viral large T (LT) antigen in MCC cells. We investigated and characterized MCPyV molecular features in MCC, respiratory, urine and blood samples from 33 patients by quantitative PCR, sequencing and detection of integrated viral DNA. We examined associations between either MCPyV viral load in primary MCC or MCPyV DNAemia and survival. Results were interpreted with respect to the viral molecular signature in each compartment. Patients with MCC containing more than 1 viral genome copy per cell had a longer period in complete remission than patients with less than 1 copy per cell (34 vs 10 months, P = 0.037). Peripheral blood mononuclear cells (PBMC) contained MCPyV more frequently in patients sampled with disease than in patients in complete remission (60% vs 11%, P = 0.00083). Moreover, the detection of MCPyV in at least one PBMC sample during follow-up was associated with a shorter overall survival (P = 0.003). Sequencing of viral DNA from MCC and non MCC samples characterized common single nucleotide polymorphisms defining 8 patient specific strains. However, specific molecular signatures truncating MCPyV LT were observed in 8/12 MCC cases but not in respiratory and urinary samples from 15 patients. New integration sites were identified in 4 MCC cases. Finally, mutated-integrated forms of MCPyV were detected in PBMC of two patients with disseminated MCC disease, indicating circulation of metastatic cells. We conclude that MCPyV molecular features in primary MCC tumour and PBMC may help to predict the course of the disease.