BCL6 has emerged as a major therapeutic target in B cell lymphomas. The safety and efficacy of the specific BCL6-targeted therapy drug RI-BPI in preclinical studies supports the use of this agent to anchor combinatorial targeted therapy. Rational design of such combinations requires knowledge of biochemical and biological mechanisms through which BCL6 maintains lymphoma cell survival. We approached this challenge by first identifying a gene signature uniformly induced by RI-BPI across BCL6-dependent DLBCL cells regardless of their genetic background. The goal of this experiment was to identify a core gene set rather than to comprehensively catalog every transcript that can be induced by RI-BPI. We reasoned that a robust core signature would be most representative of the actions of RI-BPI and thus most suitable for connectivity mapping with other drug signatures contained within the C-map database. This procedure identified HDAC and Hsp90 inhibitors as displaying partially overlapping signatures with RI-BPI. Both of these classes of drugs are pleiotropic in their actions since they can alter the acetylation and stability of numerous protein substrates respectively.
HDAC inhibitors exhibit activity against several types of B cell lymphoma cell lines in vitro and in vivo (27
), presumably due to acetylation of a diverse set of nuclear and cytoplasmatic proteins. One recent report showed that SAHA and other HDIs could upregulate up to 10% of all protein acetylation sites by at least a factor of 2 (18
). The multiplicity of protein targets coupled with the molecular heterogeneity of DLBCL make it difficult to predict which actions of HDI will predominate in a given tumor’s genetic background. Accordingly, we have not been able to identify any single genetic or biological feature among DLBCL cell lines that correlates with resistance or sensitivity to HDI (data not shown). Moreover, a phase II clinical trial with the HDAC inhibitor SAHA in relapsed DLBCL patients showed limited activity of this drug as single agent (28
). In the setting of DLBCL, this class of drugs will most likely be useful in rational combination with other agents. Hsp90 is highly expressed in DLBCL (7
) and often displays aberrant distribution with prominent nuclear localization in addition to its usual cytoplasmatic pattern (7
). In addition to maintaining the stability of oncogenic signaling proteins such as RAF1 and AKT1 (30
), Hsp90 has been shown to chaperone mutant oncogenes, including BCR-ABL in chronic myeloid leukemias (31
) and NPM-ALK in anaplastic large-cell lymphomas (34
). Hsp90-mediated stabilization of AKT has been implicated as contributing to the survival of DLBCL cells, and the Hsp90 inhibitor IPI-504 was shown to exhibit synergy with the AKT inhibitor LY24009 (35
). Of particular significance to DLBCL pathogenesis, Hsp90 mediates the stabilization of BCL6
mRNA and protein (7
). The importance of BCL6 as a substrate protein is underlined by the fact that a degradation-resistant BCL6 mutant partially rescued DLBCL cells from Hsp90 inhibitor–induced apoptosis (7
). Moreover, BCL6-independent DLBCL cells are relatively less sensitive to these drugs (7
In seeking to determine the mechanistic basis for the connectivity among RI-BPI, HDIs, and Hsp90 inhibitors, we identified EP300
as a crucial BCL6 target gene. RI-BPI blockade of BCL6 induced EP300
mRNA and protein expression as well as its KAT enzymatic activity. The importance of p300 KAT activity downstream of RI-BPI is underlined by the observations that (a) specific p300 KAT domain inhibitors or transfection of p300 dominant negative mutants (in which the KAT domain has been deleted) partially rescued the actions of RI-BPI; (b) BCL6 also directly represses BAT3, which is required for p300-mediated acetylation of target proteins, and BAT3 depletion also rescued DLBCL cells from RI-BPI; and (c) somatic p300 mutations that affect the KAT domain were observed to occur naturally in DLBCL patients, consistent with a tumor-suppressor role for this protein. Since p300-mediated acetylation of Hsp90 attenuates its chaperone functions (14
), it is reasonable to consider that BCL6 blockade could lead to reduction in Hsp90 activity. This effect would be conceptually similar to the observation that HDIs attenuate Hsp90 activity by promoting its acetylation, thus providing a functional link among these 3 classes of drugs (14
). Accordingly, we observed that in DLBCL cells, both HDI and RI-BPI induce Hsp90 hyperacetylation and a reduction in its canonical substrate proteins AKT1 and RAF1, as well as the expected compensatory increase in Hsp70 (36
). The data suggest the existence of a feedback loop between BCL6 and Hsp90, whereby BCL6 induces Hsp90 activity by suppressing its acetylation (via p300 repression) and Hsp90 sustains BCL6 activity by maintaining its mRNA and protein levels. Blockade of either arm (by either RI-BPI or Hsp90 inhibitor) disrupts both BCL6 and Hsp90 functions and leads to killing of lymphoma cells.
RI-BPI induction of p300 likely delineates additional functional links with HDI. For example, p300 acetylation of p53 can increase its transcriptional and biological actions (37
). While p53 is also a BCL6 target gene, it has been shown that not only mutant but also wild-type p53 can be expressed in DLBCL along with BCL6 (41
). Yet even though expressed, p53 function is attenuated in DLBCL cells. Along these lines, previous data indicate that RI-BPI can induce the functional activity of already expressed p53 in DLBCL cells, regardless of whether p53 was mutant or wild type (41
). p53-activating peptides or small molecules accordingly enhanced the cell-killing activity of RI-BPI, while in contrast, dominant negative p53 or the p53 inhibitor pifithrin-α partially rescued DLBCL cells from RI-BPI (41
). Connecting these data together suggests a scenario whereby RI-BPI induced, p300-mediated acetylation of p53 plays a crucial role in inducing p53 function and mediating antilymphoma effects.
Our data point toward a role for p300 as a tumor suppressor. A recent study showed that EP300
is mutated in RC-K8 DLBCL cells, resulting in loss of its KAT domain (23
). We now describe point mutations that disrupt the KAT domain of p300 in DLBCL patients as well as in several additional DLBCL cell lines. The functional impact of these mutations is highlighted by the fact that transfection of wild-type p300 in RC-K8 cells and Karpas422 DLBCL cells sensitizes these cells to RI-BPI and SAHA (but not PU-H71, which does not target protein acetylation). Loss of function mutations of the KAT domain of p300 were also reported to induce increased proliferative potential in hematopoietic progenitor cells (43
). Moreover, transfection of a dominant negative KAT mutant p300 attenuated the response of DLBCL cells to RI-BPI, similar to the effect of administering specific p300 inhibitor drugs. These results do not rule out the possibility that other proteins with KAT activity could also contribute to the effect of RI-BPI. In this regard, we found that RI-BPI could increase the mRNA levels of the CBP lysine acetyltransferase (CREB-binding protein), although to a lesser level than EP300
(data not shown). Notably, despite their similarities, p300 and CBP may regulate different sets of genes and mediate different biological effects (44
), such as those involved in the apoptotic response to DNA damage (39
). Notably, in a similar fashion to EP300
, several other BCL6 target genes with tumor suppressor activity are mutated or deleted in DLBCL including PRDM1
), and CDKN2A
). Although BCL6 represses these genes, genetic lesions would presumably more profoundly and stably inactivate these loci and alter the physiology of malignant lymphoma cells.
From the translational perspective, we predicted that RI-BPI might synergize with HDI or Hsp90 inhibitors to kill lymphoma cells by more potently inducing protein acetylation and inhibiting Hsp90. Along these lines, we observed that the combination of RI-BPI with Hsp90 inhibitors or HDI leads to a greater reduction of Hsp90 client proteins than single drug treatment and that combination of RI-BPI and HDI induces greater p300 KAT activity and histone acetylation than either agent alone. Dose-response experiments combining RI-BPI with chemically distinct HDI and Hsp90 inhibitors accordingly revealed synergy in a majority of DLBCL cell lines. While these data do not rule out that other nonoverlapping effects of these classes of drugs contribute to synergistic antilymphoma activity, they do support the rationale for combinatorial therapy. When tested in a preclinical model, these same combinations again yielded more potent suppression of lymphomas in vivo than any of the drugs alone, including complete regression of tumors in mice treated with the combination of RI-BPI and Hsp90 inhibitor. These data are potentially clinically relevant. Combinatorial therapy also displayed enhanced killing of purified primary human DLBCL cells. One of the HDAC inhibitors used in this study, SAHA, is of clinical use. The PU-H71 Hsp90 inhibitor is promising from the clinical standpoint, since it has a wider therapeutic window than most other chemical scaffolds targeting Hsp90, making it possible to deliver a more potent antitumor effect with less toxicity (7
). The fact that these drugs do not have known overlapping toxicities and were accordingly nontoxic when administered as combinations in mice further merits their consideration for use in rationally designed combinatorial targeted therapy clinical trials for patients with DLBCLs.