Development of a novel BCL6 PROTAC to provide insight into small molecule targeting of BCL6

B-cell lymphoma 6 (BCL6) inhibition is a promising mechanism for treating hematological cancers but high quality chemical probes are necessary to evaluate its therapeutic potential. Here we report potent BCL6 inhibitors that demonstrate cellular target engagement and exhibit exquisite selectivity for BCL6 based on mass spectrometry analyses following chemical proteomic pulldown. Importantly, a proteolysis-targeting chimera (PROTAC) was also developed and shown to significantly degrade BCL6 in a number of McCoull et al, ACS CB Page 2 of 31 diffuse large B-cell lymphoma (DLBCL) cell lines, but neither BCL6 inhibition nor degradation selectively induced marked phenotypic response. To investigate, we monitored PROTAC directed BCL6 degradation in DLBCL OCI-Ly1 cells by immunofluorescence and discovered a residual BCL6 population. Analysis of sub-cellular fractions also showed incomplete BCL6 degradation in all fractions despite having measurable PROTAC concentrations, together providing a rationale for the weak antiproliferative response seen with both BCL6 inhibitor and degrader. In summary, we have developed potent and selective BCL6 inhibitors and a BCL6 PROTAC that effectively degraded BCL6, but both modalities failed to induce a significant phenotypic response in DLBCL despite achieving cellular concentrations. The transcriptional repressor B-cell lymphoma 6 (BCL6) is required for germinal center (GC) formation and maintenance during the humoral immune response but has also been implicated to play a role in lymphomas derived from GC B-cells. Given the role of BCL6 in blocking cell cycle progression and attenuating DNA damage response to promote antibody affinity maturation in antigen activated B-cells, it is plausible to hypothesize that deregulating BCL6 would lead to maintenance of a highly proliferative GC phenotype with accumulating DNA damage, eventually leading to malignant transformation. Indeed, BCL6 was found to be deregulated in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma cancers through a number of genetic alterations 3 and its oncogenic potential was validated in transgenic mice constitutively expressing IμHA-BCL6 eventually developing lethal DLBCL-like tumours. Furthermore, BCL6 dependency in the disease setting was demonstrated through BCL6 shRNA-induced lethality in DLBCL cell lines, while small molecule BCL6 inhibitors based on a promiscuous inhibitor class and peptido-mimetic inhibitors have been reported to lead to an anti-proliferative phenotype in DLBCL. McCoull et al, ACS CB Page 3 of 31 Consequently, BCL6 has been proposed as an attractive cancer therapeutic target but as many cancer targets do not fulfill the promise of their preclinical data, there is a need for potent and selective tools to improve understanding of the BCL6 inhibition phenotype in DLBCL. The protein-protein interaction (PPI) between a homodimer of the N-terminal broadcomplex, tramtrack and bric a brac (BTB) domain of BCL6 and corepressors silencing mediator for retinoid or thyroid-hormone receptors (SMRT), nuclear receptor corepressor (NCOR) and BCL6 corepressor (BCOR), 10 leads to the repression of genes through which the survival and proliferation effects of GC B-cells is allowed to progress. Within medicinal chemistry, PPI inhibition provides a significant challenge, potentially explaining the lack of BCL6 inhibitors published to date. Recently, a high affinity BCL6-binding peptide has been reported, as well as a fragment-screen derived small molecule binder. We also recently reported high affinity, selective small molecule cellular probes for BCL6 but they failed to demonstrate a significant BCL6 dependent antiproliferative effect in DLBCL cell lines. Here, we present a new series of high activity, selective small molecule BCL6 inhibitors which we were also able to convert into chemical biology tools. Cellular target engagement was confirmed through BCL6 thermal stabilization (CETSA) and further demonstrated through BCL6 specific degradation in a number of cell lines with a PROTAC molecule, but neither the BCL6 inhibitor nor degrader showed improved or selective antiproliferative response compared to our previously reported inhibitors. Through additional investigation, we showed by immunofluorescence imaging and sub-cellular fractionation studies that the degradation of BCL6 with PROTAC appears incomplete, as a residual population remains despite measurable PROTAC concentrations. McCoull et al, ACS CB Page 4 of 31 RESULTS AND DISCUSSION Developing PROTAC ready BCL6 ligands. To design small molecule ligands suitable for converting to effective PROTACs, we considered high permeability to be a primary requirement in addition to cellular potency and selectivity. Our previous Pyrazolo[1,5a]Pyrimidine (PP) series of BCL6 binders lacked sufficient permeability and thus a new 5Chloro-Pyrimidine (CP) chemical series was evolved from fragment screening hit 1 (Figure 1A). A similar fragment hit has recently been reported. 2D NMR analysis confirmed that triazine 1 bound BCL6 competitively with its corepressor SMRT and a crystal structure further confirmed the position of the ligand along the lateral groove formed between both chains of the homodimer (Figure 1B). Screening of structural near neighbors of 1 identified the CP 2 with greater than 100-fold activity increase which was also confirmed to bind in the lateral groove in a crystal structure (Figure 1C). The fluorine is directed towards, but does not contact, Glu115 thus a lactam group was added to make a hydrogen bonding interaction with Glu115 and this gave a 12-fold increase in activity in 3. Such 2-NH-pyrimidine motifs are precedented in kinase inhibitors 18 and since this NH does not form an obvious interaction with BCL6 protein on binding, it was removed through incorporation into a morpholine ring in 4 to maintain BCL6 activity but reduce kinase inhibitory activity (Table S1). The bound conformation of 4 was not the lowest energy conformation of the isolated ligand and the free-ligand conformational analysis by NMR showed that the bound conformation of 4 is only 50% populated (Figure S1A). Consequently, macrocyclization to 5 was predicted to favour the bioactive conformation and this achieved a further 5-fold activity increase. Free-ligand analysis of macrocycle 5 shows a pattern of NMR signals indicative of a rigid linker and a single 100% populated conformer in solution. Finally, switching to piperazine 6 allowed the opportunity to convert either of the NMe groups to a linker for McCoull et al, ACS CB Page 5 of 31 attachment of probe moieties, and the addition of a fluorine in the 6-position of the pyrimidine was tolerated for potency.