The expanding role of the BCL6 oncoprotein as a cancer therapeutic target

Abstract

BCL6 was initially discovered as an oncogene in B-cell lymphomas, where it drives the malignant phenotype by repressing proliferation and DNA damage checkpoints and blocking B-cell terminal differentiation. BCL6 mediates its effects by binding to hundreds of target genes, and then repressing these genes by recruiting several different chromatin modifying corepressor complexes. Structural characterization of BCL6-corepressor complexes suggested that BCL6 might be a druggable target. Accordingly a number of compounds have been designed to bind to BCL6 and block corepressor recruitment. These compounds, based on peptide or small molecule scaffolds, can potently block BCL6 repression of target genes and kill lymphoma cells. In the case of diffuse large B-cell lymphomas (DLBCLs), BCL6 inhibitors are equally effective in suppressing both the GCB- and the more aggressive ABC-DLBCL subtypes, both of which require BCL6 to maintain their survival. In addition, BCL6 is implicated in an expanding scope of hematologic and solid tumors. These include but are not limited to B-acute lymphoblastic leukemia, chronic myeloid leukemia, breast cancer and non-small cell lung cancer. BCL6 inhibitors have been shown to exert potent effects against these tumor types. Moreover mechanism based combinations of BCL6 inhibitors with other agents has yielded synergistic and often quite dramatic activity. Hence there is a compelling case to accelerate development of BCL6 targeted therapies for translation to the clinical setting.

INTRODUCTION

BCL6 (B-cell lymphoma 6) is emerging as a key oncoprotein and therapeutic target. BCL6 was first identified as a locus affected by chromosomal translocations in diffuse large B-cell lymphomas (DLBCLs) (1). However it is now known to be broadly expressed in many lymphomas regardless of genetic lesions. Its role in lymphomagenesis stems from its function in the humoral immune system, where upregulation of BCL6 is required for the formation of germinal centers (GCs) during the humoral immune response (2–4). GCs are transient structures that form in response to antigen stimulation. Within GCs B-cells tolerate massive proliferation and the mutagenic effect of the DNA editing enzyme AICDA in order to undergo immunoglobulin affinity maturation (5). All of this is orchestrated by and dependent on BCL6, a powerful transcriptional repressor that silences hundreds of genes. Some of these control DNA damage sensing (i.e. ATR, CHEK1, TP53, ARF, etc), and proliferation checkpoints (CDKN1A, CDKN1B, CDKN2A, CDKN2B, PTEN, etc. (6). BCL6 also represses genes required for exit from the GC reaction and plasma cell differentiation (e.g. IRF4, PRDM1) (6). This ensures that GC B-cells have sufficient time to acquire somatic hypermutation of their immunoglobulin genes. It thus is easy to visualize how deregulated suppression of these target genes could result in malignant transformation of B-cells. Indeed constitutive expression of BCL6 in GC B-cells drives the development of DLBCL in mice (7–9).

BCL6 also represses numerous oncogenes in GC B-cells, including MYC, BCL2, BMI1, CCND1 and various others (10, 11). Through this function BCL6 may mitigate its own pro-oncogenic checkpoint repression effect and thus reduce the potential for malignant transformation of GC B-cells. This effect is abrogated in the presence of BCL2 or MYC translocations, which drive expression of these oncogenes through aberrant regulatory elements. The presence of both MYC and/or BCL2 together with BCL6 (regardless of translocations) is clearly deleterious. It provides B-cells with simultaneous suppression of checkpoints through BCL6 along with the pro-growth and survival effects of MYC and BCL6. Not surprisingly the combination of MYC and/or BCL2 with BCL6 in DLBCL has been linked to unfavorable clinical outcomes (12).

In the normal immune response BCL6 function is terminated by disruption of BCL6 transcriptional complexes through CD40 induced ERK signaling, and downregulation of BCL6 mRNA by IRF4 and PRDM1 (13–15). Termination of BCL6 function is required for B-cells to exit the GC reaction. Yet in DLBCLs a variety of mechanisms contribute to aberrant persistence of BCL6 expression. These include fusion of the BCL6 coding region to heterologous promoters via chromosomal translocations and somatic mutation of binding sites for repressors of BCL6 expression such as IRF4, and BCL6 itself (15, 16). Somatic mutations of the BCL6 ubiquitin ligase FBXO11 can enhance the half-life of BCL6 protein in DLBCL (17). Induction of Hsp90 activation which occurs almost universally in DLBCL forms a positive feedback loop whereby i) HSP90 maintains BCL6 mRNA and protein stability and ii) enhances BCL6 repressor function by directly forming a complex on chromatin; iii) BCL6 repression of EP300 prevents acetylation and inactivation of HSP90, thus further enhancing BCL6 protein expression (18, 19). BCL6 expression can also be aberrantly maintained by hypermethylation of regulatory CpGs contained in the BCL6 first intron (20). The powerful tumorigenic activity of BCL6 and the myriad ways that lymphoma cells maintain its activity have fueled interest in development of BCL6 inhibitors.

The biological functions of BCL6 are mediated through distinct and specific structural motifs

In order to understand BCL6 as a therapeutic target it is first necessary to consider how it mediates its biological actions. BCL6 has a trimodular structure consisting of an N-terminal BTB/POZ domain that mediates transcriptional repression, an unstructured middle region containing a second repression domain (RD2), and a series of six C2H2 zinc fingers at the C-terminus that bind to DNA and other proteins (6). The biochemical and biological functions, together with the partner proteins of each of BCL6 domains are summarized in Figure 1. One approach to targeting BCL6 is to completely abrogate its functions, for example using molecules that could block its zinc fingers from binding to DNA, or by destroying or downregulating the entire protein using approaches such as RNAi, antisense molecules, or small molecules that target proteins for proteolytic destruction (e.g. degronomids). Yet in the case of BCL6 such an effect is not desirable. This is because BCL6 knockout mice in addition to failure to form GCs, also manifest a severe and lethal systemic inflammatory disease driven by T-cells and macrophages (21). BCL6 deficient mice die within a few weeks of birth and exhibit massive tissue infiltration of inflammatory cells in lung, heart and other tissues (2–4). Loss of BCL6 in macrophages causes accelerated atherosclerosis in mice (22).

Figure 1. The biological functions of BCL6 are mediated through specific protein domains.

The figure shows a cartoon representation of BCL6 domain structure indicating for each one the biochemical function, partner proteins, and biological functions.

Fortunately it is possible to avoid these adverse effects by targeting specific sites on BCL6 that drive its cancer functions but leave its anti-inflammatory functions intact. Structure function studies show that the BCL6 BTB domain mediates repression by recruiting the corepressor proteins SMRT, NCOR and BCOR to an extended groove motif that forms along the BTB dimer interface (Figure 1 and Figure 2A and B) (23, 24). BCL6 can bind two corepressors at a time because there are grooves on either side of the dimer. The corepressors bind through an 18 amino acid BCL6 binding domain (BBD) that is highly conserved between SMRT and NCOR but which is completely different in the case of BCOR (23, 24). Importantly the BCL6 lateral groove residues that make contact with the BBD are unique to BCL6 and not other BTB proteins (23), which could facilitate development of specific inhibitors that do not affect other BTB domains. Point mutation of the lateral groove abolishes the repressor function of the BCL6 BTB domain (23).

Figure 2. The BTB domain of BCL6.

The images represent the BCL6 BTB domain alone or in a complex with the SMRT BBD or compound 79-6 as determined by X-ray crystallography. The BTB domain forms an obligate homodimer, with the two monomers shown in red and blue in each panel. A) Ribbon view, with arrows pointing to the charged pocket motif and the hydrophobic surface, indicating examples of specific residues that participate in these features. B) Space fill representation of the BCL6 BTB domain in a complex with the SMRT BBD, which is the polypeptide chain shown in purple. The lateral groove is indicated in the boxed area.

The repressive mechanism of action of the BCL6 BTB domain is quite intricate. NCOR and SMRT have related structures and form similar chromatin modifying complexes that deacetylate histones (25, 26), whereas BCOR is completely different and forms a variant Polycomb PRC1 complex with multiple distinct effects on chromatin (27). On the one hand BCL6 preferentially represses gene promoters by forming a complex with BCOR (28). However, the BCOR complex can only stably assemble at promoters that are marked with histone 3 lysine 27 trimethylation (H3K27me3) by the Polycomb PRC2 protein EZH2 (29). H3K27me3 forms a binding site for CBX8, one of the components of the BCOR complex. Hence BCL6 mediated promoter repression in GC and lymphoma cells requires “combinatorial tethering” of BCOR complex through the simultaneous actions of BCL6 and EZH2 (29). On the other hand BCL6 also represses gene enhancers, but this effect is linked to preferential interaction with SMRT and NCOR complexes, which include HDAC3 (28). BCL6 enhancer repression is driven through this indirect recruitment of HDAC3 to remove the activating H3K27acetyl mark. Notably the genes that BCL6 represses by forming promoter repression complexes are mostly different from those that it represses by forming enhancer complexes (28).

Remarkably, mice expressing a mutated form of BCL6 where the BTB domain cannot bind to SMRT, NCOR and BCOR live normal healthy lives without any evidence of inflammatory syndrome. These mice are nonetheless unable to form GCs, specifically due to failure of GC B-cells to proliferate and survive (30). This is because BCL6 largely represses DNA damage and proliferation checkpoint genes through the BTB domain, which may explains (as discussed below) why this domain is also important in other tumor types. Indeed mutation of the BTB domain abrogated the ability of the lymphoma oncogene EZH2^Y641 to drive preneoplastic lymphoproliferation in vivo (29). Hence the BCL6 BTB domain is required for GC formation and lymphomagenesis but not anti-inflammatory effects. Instead the anti-inflammatory effect is due to direct competition of the BCL6 zinc fingers with STAT proteins for binding to inflammatory chemokines in macrophages (30). Finally, the minimal BCL6 RD2 domain consists of ~40 amino acids and is required for interactions with MTA3 and HDAC2 (31, 32). Mice engineered to express RD2 mutant BCL6 also live normal healthy lives and have no inflammation (32). They do lack GCs, although in this case the mechanism involves partial loss of function of T-follicular helper (TFH) cells, and disruption of B-cell homing to the GC (32). This extraordinary cell context biochemical specialization of BCL6 makes it possible to target specific functions relevant to tumor cells and at the same time avoid disrupting other critical functions.

Development of peptidomimetic BCL6 inhibitors

The most straightforward approach to targeting BCL6 is disrupting the interaction between BCL6 BTB domain lateral groove and its corepressors because i) this protein surface mediates BCL6 oncogenic effects, ii) the protein interaction is structurally characterized, iii) it involves a unique interface not conserved in other proteins, and iv) targeting the BTB lateral groove will not induce deleterious inflammatory effects caused by complete loss of BCL6. Targeting the BTB dimer interface would likely be unsuitable since BTB domains aggregate and are degraded if they cannot dimerize, which could unleash the inflammatory effects of BCL6 deficiency. The BTB domain has other features such as a charged pocked motif and a hydrophobic surface for oligomerization, which have not been functionally characterized (Figure 2A) (23).

Protein-protein interactions have long been thought to be “undruggable” (33), given that many protein interactions involve large surfaces that may be difficult to disrupt with small molecules. However given sufficient knowledge of the protein interface it is proving possible to design drugs that exploit vulnerable “sweet spots” (34). The first BCL6 inhibitor contained the 17 amino acids from SMRT-BBD along with a protein transduction domain (PTD) for cell penetration (35). This BCL6 peptide inhibitor (BPI) disrupted BCL6 repression complexes, induced expression of BCL6 target genes, killed DLBCL cells in vitro and phenocopied the BCL6 BTB domain mutant phenotype in vivo (i.e. loss of GC formation without inflammation) (Figure 3). Although BPI was active at low micromolar concentrations, it was also readily degraded by proteases (35). The bulk of intermolecular contacts between the BBD and BCL6 are confined to a 9 amino acid sequence, which was used as the warhead for a second-generation peptide using D-amino acids resistant to proteases (36). The peptide sequence was inverted to maintain proper stereochemistry and included a fusogenic motif for superior uptake within cells. The resulting retro-inverso (RI)-BPI peptide retained specificity for BCL6 but not other BTB proteins, exhibited superior stability and did not induce immunogenicity (36). RI-BPI killed BCL6-dependent DLBCLs at an average concentration of 16 µM, similar to BPI (Figure 3) (36). Pharmacokinetic studies in mice revealed peak RI-BPI intra-tumor concentrations after 30 minutes and persistence in the nuclei of lymphoma cells for >24 hours (36). RI-BPI was non-toxic to mice even after one year of continuous administration (36). These peptides thus appear suitable for use in humans. An alternative peptidomimetic strategy employed aptamer screening to identify Apt48, which binds to the BCL6-BTB domain in a different manner than BPI (Figure 3) (37). Apt48 promoted re-expression of BCL6 target genes and cell cycle arrest in lymphoma cells, suggesting interference with BCL6 function.

Figure 3. Current BCL6 inhibitors.

The table depicts the structure and known activities of published BCL6 inhibitors. Abbreviations: NA, not applicable; SPR, surface plasmon resonance; ITC, isothermal titration calorimetry; EMSA, electrophoretic mobility shift assay; NMR, nuclear magnetic resonance; MST, MicroScale Thermophoresis; qPCR quantitative PCR; ChIP, Chomatin immunoprecipitation; GI₅₀, growth inhibition 50%.

BCL6 small molecule inhibitors

The BCL6-BTB lateral groove-BBD complex was used for in silico screening of small molecule libraries. These studies yielded a family of compounds with reproducible and specific BCL6 inhibition. A representative compound called 79-6 was shown to bind an aromatic pocket situated in the BTB domain lateral groove (38). 79-6 induced disruption of BCL6 transcriptional complexes, reactivation of BCL6 target genes and selective killing of BCL6-dependent DLBCL cells (Figure 3) (38). Even though this small molecule was active in animal models, its binding affinity of ~138 µM was lower than the endogenous BBD (~20 µM) (38). A novel in silico-based fragment based screening method, called SILCS (39, 40), was subsequently used to design the most potent BCL6 inhibitor to date, called FX1 (41). FX1 is the first reported inhibitor with higher affinity for the BCL6 BTB domain than the endogenous corepressor proteins (IC50 ~ 4 µM), demonstrating the feasibility of blocking large protein interactions with small molecules (Figure 3) (41). SILCS modeling combined with NMR analysis revealed a more favorable orientation of the compound within the lateral groove aromatic pocket. FX1 outperformed all previous BCL6 inhibitors in cell-based pharmacodynamic assays, while maintaining specificity for BCL6. The molecule exhibited favorable pharmacokinetics in vivo and lack of toxicity similar to previous generations of BCL6 inhibitors (41). Other small molecules with potential BCL6 binding activity include the natural product Resveratrol (42), which can induce anti-proliferative and anti-inflammatory activities in different types of cancer cells through various mechanisms (Figure 3) (43). Ansamycin antibiotics may also bind to BCL6. The structure of one of the members of this family rifabutin, with the BCL6-BTB domain showed binding to the lateral groove similar to 79-6, although with a lower binding affinity of approximately 1 mM (Figure 3) (44).

Pre-clinical activity of BCL6 inhibitors in GCB type DLBCLs

High BCL6 expression is usually associated with GCB type DLBCLs (45), which are the most obvious target disease for BCL6 inhibitors. Accordingly daily administration of RI-BPI at a dose of 7.5 mg/kg/day reduced the growth of established BCL6-dependent GCB-DLBCL xenografts by 60–80% but did not affect BCL6 independent DLBCL xenografts (36). Activity was concentration dependent, with doses of 50mg/kg/day leading to complete regression of GCB-DLBCLs (6). In spite of its weak binding the small molecule 79-6 concentrates to high levels within lymphoma cells, which enhances its activity (38). Administration of 50 mg/kg/day 79-6 to SCID mice induced a 65–70% reduction in the size of established BCL6-dependent DLBCL xenografts, but had no effect on BCL6-independent DLBCLs (38). In contrast the potent small molecule FX1 induced complete regression of GCB-DLBCL xenografts at 25 mg/kg, but did not suppress BCL6 independent lymphomas (41). In all of these studies the various inhibitors induced BCL6 target gene re-expression, proliferation arrest and apoptosis in DLBCL tumor xenografts. More importantly, all three compounds were active in killing primary human DLBCL specimens cultured ex vivo underlining the potential for BCL6 inhibitors to have activity clinical trials (36, 38, 41).

From the predictive biomarker perspective, it would be useful to classify patients as having BCL6 dependent or BCL6 independent lymphomas. The simple expression of BCL6 may not be sufficient to indicate BCL6 dependence since a few GCB-DLBCL cell lines express BCL6 but are not affected by genetic or pharmacological BCL6 inhibition (36, 38, 41, 46). A recent study overexpressing BCL6 in hematopoietic cells in mice showed development of lymphomas that were BCL6 independent. These data suggest that BCL6 might sometimes act in a hit-and-run manner (47). Expression of BCL6 is therefore necessary but perhaps not in all cases sufficient to predict response to BCL6 inhibitors. Gene expression profiles might help to identify BCL6 dependent lymphomas. Indeed one study identified a putative BCL6 functional signature by analyzing transcriptional profiles in primary DLBCL patient specimens. When applied to DLBCL cell lines this signature accurately predicted which ones would be BCL6 dependent and which ones are BCL6 independent based on their response to BCL6 inhibitor (46). Perhaps functionally relevant biomarkers such as these could aid in patient selection for BCL6 inhibitor trials.

Which lymphomas can be targeted by BCL6 inhibitors?

The relevance of BCL6 is not limited to GCB-DLBCLs. In fact, BCL6 is expressed in most ABC-type DLBCLs, albeit at lower levels (41, 48). Indeed BCL6 translocation and amplification is largely restricted to ABC-DLBCLs providing genetic evidence for the importance of BCL6 in these tumors (47). It is worth noting that BCL6 transcript or protein abundance is not associated with degree of biological dependency on BCL6. Along these lines BCL6 shRNA knockdown of BCL6 was just as deleterious to ABC-DLBCL cells as to GCB-DLBCL cells (41). ABC-DLBCL cells respond to similar doses of BCL6 inhibitor such as FX1, which also suppressed the growth of ABC-DLBCL xenografts in vivo. Most importantly, primary human ABC and GCB-DLBCL cells respond equally well to FX1 ex vivo (41). Given that ABC-DLBCLs manifest inferior outcome, it is warranted to develop BCL6 targeted therapy for these patients. Follicular lymphomas manifest features of germinal center B cells and thus generally express BCL6. We have found that primary low-grade follicular lymphoma cells are killed by the BCL6 inhibitor RI-BPI (E Valls and A Melnick, unpublished data). Burkitt lymphomas arise from GC B-cells, and BL cell lines are potently killed by BCL6 inhibitors in vitro and in vivo (35). Although BCL6 is not usually expressed in multiple myeloma, withdrawal of microenvironment survival signals could induce BCL6 expression (49), raising the possibility that these B-cell neoplasms could be sensitive to BCL6 inhibitors as part of combination therapy regimens.

Therapeutic targeting of BCL6 in other tumors

BCL6 is implicated in an expanding list of tumors. Table 1 summarizes the spectrum of BCL6 dependent tumors, the way in which BCL6 was inhibited and the phenotype associated with the inhibition. For example, BCL6 is a direct transcriptional target of MLL fusion proteins in 11q23 rearranged B-acute lymphoblastic leukemias (B-ALL) (50). Binding of MLL fusion protein to the BCL6 promoter was associated with hypomethylation and induction of BCL6 expression. Treatment with RI-BPI reduced colony formation capacity and induced apoptosis in MLL fusion cell lines and primary patient samples (50). Exposure to ABL tyrosine kinase inhibitors (TKI) strongly induces BCL6 expression in BCR-ABL+ B-ALLs. In this context BCL6 functioned as a survival feedback mechanism that enables leukemia cells to resist TKI treatment (51). Combined treatment of primary human BCR-ABL+ B-ALL with TKI and RI-BPI yielded massively synergistic anti-leukemia effects in vitro and in vivo (51). Chronic myeloid leukemias are also driven by BCR-ABL fusion protein. Similar to B-ALLs, exposure to TKI induced BCL6 in CML cells (52). CD34+ CML leukemic stem cells (LSCs) upregulate BCL6 in response to TKI in a FOXO-dependent manner. It has been appreciated that CML LSCs are less responsive to TKI than bulk leukemia cells and in many patients the disease is not eradicated by TKI therapy alone (52). It is notable that CML LSCs require BCL6 for their ability to form colonies, and initiate leukemia in mice. Treatment of patient-derived CML cells with RI-BPI selectively induced cell cycle arrest of CD34+ LSCs causing depletion of CD34+ CD38- LSCs whereas CD34− subpopulations remained intact (52). Hence BCL6 targeted therapy could potentially eradicate CML LSCs and reduce the need for long-term TKI therapy.

Table 1.

Therapeutic targeting of BCL6 in lymphomas and other tumors.

Malignancy		Description	BCL6 RNA	BCL6 inhibiton	Phenotype	In vivo inhibition	Reference
Lymphoma	GCB- DLBCL	Germinal center B-cell Diffuse Large B-cell Lymphoma	Yes	siBCL6/shBCL6/BPI/RI- BPI/79-6/FX1	Decreased proliferation and apoptosis	Yes	21, 29, 31, 34
	ABC- DLBCL	Activated B-cell Diffuse Large B-cell Lymphoma	Yes	siBCL6/shBCL6/BPI/RI- BPI/79-6/FX1	Decreased proliferation and apoptosis	Yes	21, 29, 31, 34
	FL	Follicular Lymphoma	Yes	RI-BPI	Apoptosis	NA	51
Ph+ B-ALL		B-cell acute lymphoblastic leukemia with Philadelphia chromosome	Yes	shBCL6/RI-BPI	Reduced colony formation and delayed progression	Yes	44
MLLr B-ALL		MLL rearranged B-cell acute lymphoblastic leukemia	Yes	siBCL6/RI-BPI	Apoptosis	NA	43
CML		Chronic Myeloid Leukemia	Yes	RI-BPI/dnBCL6	Selectively eradicates CD34+ CD38- leukemia- forming colonies	Yes	45
Breast Cancer		Breast Cancer	Yes	siBCL6/79-6	Reduced EMT and invasion	Yes	46, 47, 48
NSCLC		Non-Small Cell Lung Cancer	Yes	FX1/shBCL6	Inhibits proliferation	Yes	49

The spectrum of tumors that are dependent on BCL6 and are suppressed by BCL6 inhibitors. NA, not applicable.

Angioimmunoblastic T-cell lymphomas (AITL) manifest the phenotypic hallmarks of TFH cells. BCL6 is required for the formation of TFH cells (53). AITLs strongly express BCL6 and hence it has been proposed that BCL6 might be a therapeutic target in this disease (53). Up to 50% of breast tumors and many breast cancer cell lines contain amplification of the BCL6 locus (54–56). Functional studies suggest that BCL6 contributes to development of breast cancer (55–58). BCL6 is important for tumor maintenance since treatment of breast cancer cell lines with RI-BPI or 79-6 reduced cell viability (54). BCL6 may have cell context specific functions in breast cancer cells since its target genes are only partially overlapping with those in B cells (54). BCL6 was shown to be required for survival and proliferation of NSCLC cells, in part due to repression of genes involved in the DNA damage response. Inhibition of BCL6 by FX1 kills NSCLC cells in vitro and in vivo and exerts synergistic effect with cisplatin in xenograft models (59).

Combination therapies of BCL6 with other inhibitors

Given the complexity and heterogeneity of tumor cells it is unlikely that single agents will cure disease. Hence rational combination of BCL6 inhibitors with other drugs is the best way to achieve maximal anti-tumor effect. Blocking the BCL6 BTB domain is not expected to induce major biological side effects so it should be straightforward to combine BCL6 inhibitors with other therapeutic agents (36, 38, 41). For example, the fact that BCL6 and EZH2 cooperate and require each other to repress transcription led to the notion of combining EZH2 and BCL6 inhibitors (29). As might be expected this combination yielded enhanced re-activation of BCL6/EZH2 target genes, with corresponding cooperative effects against DLBCL cells in vitro and in vivo as well as against primary human DLBCL specimens ex vivo (29). The result was observed in EZH2 mutant and wild type DLBCL cells and is summarized in Figure 4A (29).

Figure 4. Strategies for rational combinatorial therapy against BCL6 in lymphomas.

A) Targeting the BCL6-EZH2 combinatorial tethering mechanism. BCL6 and EZH2 cooperate to recruit the BCOR corepressor complex. BCL6 directly binds to BCOR and EZH2 deposits the H3K27me3 mark that is bound by the CBX8 subunit of the BCOR complex. Targeting BCL6 and EZH2 together results in more thorough disabling of transcriptional repression in lymphomas and greater therapeutic effect. B) Targeting the HSP90-BCL6 positive feedback loop through which BCL6 targets including TP53 are suppressed at the transcriptional and post-translational levels. Disruption of this axis can be partially achieved by targeting BCL6, HSP90 or HDACs, but more complete suppression of this axis by hitting at least two of these components is highly synergistic. C) Targeting the BCL6-BCL2 oncogene switching mechanism. Targeting BCL6 results in derepression of BCL2, enabling lymphoma cells to survive by switching to a dependency on BCL2. Targeting both together prevents lymphoma cells from utilizing this escape mechanism, and also is useful in cases with BCL2 translocation where BCL2 has become independent of regulation through BCL6.

The positive feedback loop between BCL6, Hsp90 and EP300 mentioned earlier was the basis to combine BCL6 inhibitor with drugs against either Hsp90 or HDAC inhibitors (18). This is because inhibition of Hsp90 impairs BCL6 repressor functions and reduces BCL6 mRNA and protein levels (Figure 4B). This approach yields enhanced derepression of BCL6 target genes such as EP300, resulting in reduced Hsp90 activity through EP300 mediated acetylation, increased TP53 expression and function, and restored DNA damage and proliferation checkpoints (18). Accordingly, the combination of RI-BPI with Hsp90 or HDAC inhibitor was highly synergistic in vitro and yielded more potent anti-lymphoma effects in vivo (18). In a similar vein, enhanced checkpoint reactivation through combination of BCL6 inhibitor and TP53 activator also yielded enhanced anti-tumor effects (60). Indeed since BCL6 reactivates DNA damage checkpoints, it is not surprising that combinations of chemotherapy drugs plus BCL6 inhibitor yields enhanced activity against lymphomas and leukemias (41). The feedback mechanism where upregulation of BCL6 can protect BCR-ABL+ neoplasms from TKIs supports the testing of BCL6 inhibitor combination therapy in B-ALL and CML (51, 52).

Other combination therapies could target feedback mechanisms that enable cells to escape killing by BCL6 inhibitors. For example BCL6 directly binds and represses BCL2 and BCL2L1 (BCL-XL) (10, 11) (Figure 4C). Inhibition of BCL6 induces the expression of BCL2 and BCL-XL, which can help lymphoma cells survive exposure to BCL6 inhibitors, a phenomenon that has been called “oncogene switching”. The combination of BCL6 inhibitors with BH3 mimetics overcomes this effect and yields synergistic activity (61). BCL6 represses multiple NFkB related genes, which are induced after inhibiting BCL6. The effects of NFkB are complex, but proof of principle experiments combining proteasome inhibitors with BCL6 inhibitors showed highly synergistic actions. BCL6 also represses STAT3 (62, 63), an oncogenic driver in tumors including breast cancer, which could be induced by BCL6 inhibitors. Suppression of STAT activation through Jak2 or STATs inhibition caused additive loss of breast cancer cell viability, perhaps in part by suppressing additional STAT pro-tumorigenic effects (54, 63). As the functions of BCL6 in various tumor types are better understood it is likely that other such mechanism based opportunities will be developed.

In summary, BCL6 is a suitable therapeutic target that plays a broad and critical role in many cancers. Targeting the BCL6 BTB domain is an effective means of disrupting its functions without inducing significant off-target effects against normal tissues. Mechanism based combination therapy of BCL6 inhibitors yields dramatic anti-tumor effects. Hence we propose that development of clinical grade BCL6 compounds is a mission worth vigorously pursuing.