Inhibition of SYK or BTK augments venetoclax sensitivity in SHP1-negative/BCL-2-positive diffuse large B-cell lymphoma

Binu K. Sasi1 ● Claudio Martines1 ● Elena Xerxa1 ● Fabiola Porro1 ● Hilal Kalkan1 ● Rosa Fazio1 ● Sven Turkalj1 ●Engin Bojnik1 ● Beata Pyrzynska2 ● Joanna Stachura2 ● Abdessamad Zerrouqi2 ● Małgorzata Bobrowicz2 ●Magdalena Winiarska2 ● Valdemar Priebe3 ● Francesco Bertoni 3 ● Larry Mansouri4 ● Richard Rosenquist5 ● Dimitar G. Efremov1

Abstract

The BCL-2 inhibitor venetoclax has only limited activity in DLBCL despite frequent BCL-2 overexpression. Since constitutive activation of the B cell receptor (BCR) pathway has been reported in both ABC and GCB DLBCL, we investigated whether targeting SYK or BTK will increase sensitivity of DLBCL cells to venetoclax. We report that pharmacological inhibition of SYK or BTK synergistically enhances venetoclax sensitivity in BCL-2-positive DLBCL cell lines with an activated BCR pathway in vitro and in a xenograft model in vivo, despite the only modest direct cytotoxic effect. We further show that these sensitizing effects are associated with inhibition of the downstream PI3K/AKT pathway and changes in the expression of MCL-1, BIM, and HRK. In addition, we show that BCR-dependent GCB DLBCL cells are characterized by deficiency of the phosphatase SHP1, a key negative regulator of the BCR pathway. Re-expression of SHP1 in GCB DBLCL cells reduces SYK, BLNK, and GSK3 phosphorylation and induces corresponding changes in MCL1, BIM, and HRK expression. Together, these findings suggest that SHP1 deficiency is responsible for the constitutive activation of the BCR pathway in GCB DLBCL and identify SHP1 and BCL-2 as potential predictive markers for response to treatment with a venetoclax/BCR inhibitor combination.

Introduction

Diffuse large B cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin’s lymphoma, accounting for 30–40% of all newly diagnosed cases. It is a clinically and genetically heterogeneous disease, characterized by multiple genetic alterations. Gene expression profiling (GEP) studies have identified 2 major DLBCL subtypes, termed germinal center B cell-like (GCB) and activated B cell-like (ABC) subtype, which represent lymphomas arising from different stages of lymphoid differentiation [1]. These two subtypes also differ in terms of response to standard chemoimmunotherapy and display subtypespecific genetic alterations [2, 3].
DLBCL is readily curable with chemoimmunotherapy in the majority of patients, even in the most advanced cases. However, relatively few effective treatment options exist for patients with primary-refractory disease or patients that relapse following initial response to therapy [4]. These patients are typically treated with high-dose chemotherapy and autologous stem cell transplantation, which provide the best chance for cure. However, due to advanced age and comorbidities, only few of these patients will proceed to transplant, raising the need for alternative treatment options.
The BCL-2 inhibitor venetoclax was recently approved for the treatment of patients with relapsed chronic lymphocytic leukemia (CLL) and has also been evaluated in other B cell malignancies characterized by BCL-2 overexpression. However, the results of a recent phase I study of single-agent venetoclax in relapsed/refractory DLBCL were not very impressive, with only 18% of patients responding to treatment [5]. This low response rate was somewhat unexpected, considering that BCL-2 is overexpressed in up to 70% of DLBCL patients because of BCL-2 gene translocation, amplification, or other mechanisms [6–8]. Moreover, analysis of BCL-2 expression levels in a subset of DLBCL patients enrolled in this trial found no clear association between BCL-2 protein levels and clinical response [5], suggesting that BCL-2 expression is not the only factor that determines venetoclax sensitivity.
The BCL-2 homolog MCL-1 has been shown to confer venetoclax resistance in various hematological malignancies, including DLBCL and CLL [9–13]. This antiapoptotic protein is not inhibited by venetoclax and is frequently overexpressed in DLBCL cells [10, 14]. The mechanism(s) responsible for MCL-1 overexpression in DLBCL are still unknown, whereas in CLL its overexpression has been linked to post-transcriptional upregulation by BCR signals [15, 16].
Transcriptional profiling and functional studies have identified a subset of DLBCL tumors that are characterized by more abundant expression of proximal BCR signaling components and dependence on BCR signals for proliferation [17–20]. Such tumors have been identified among both the ABC and GCB DLBCL subtype. However, differences have been observed between the two subtypes in terms of mechanism of BCR pathway activation and activity of downstream signaling molecules. In the ABC DLBCL subtype, activation of the BCR pathway has been reported to occur because of reactivity of the tumor immunoglobulins with self-antigens located on the same or surrounding cells [21]. In approximately 50% of these tumors, this “chronic active” BCR signal is further amplified by mutations in the CD79A or CD79B subunit of the BCR, which inhibit BCR internalization and prevent recruitment of negative regulators of BCR signaling [19]. These tumors typically have high basal NF-kB and PI3K/AKT activity [21–23]. In contrast, BCR-dependent GCB DLBCL tumors have low baseline NF-kB activity and lack CD79A/CD79B mutations, suggesting a different mechanism of BCR pathway activation [20, 24].
Drugs that inhibit BCR signaling, such as the SYK inhibitor fostamatinib and the BTK inhibitor ibrutinib, have been tested in recent phase 1/2 clinical trials of relapsed/ refractory DLBCL and have shown relatively limited activity as single agents [22, 25, 26]. However, both drugs were well tolerated, suggesting that they could have a potential role in combination treatments. Based on the above, we investigated in the current study whether treatment with R406 (the active substance of fostamatinib) or ibrutinib will sensitize BCR-dependent DLBCL cell lines to venetoclax. We show that inhibition of BCR signaling induces coordinate changes in MCL-1, BIM, and HRK that sensitize DLBCL cells to venetoclax and identify loss of SHP1 as a frequent mechanism of BCR pathway activation in GCB DLBCL.

Materials and methods

DLBCL cell lines SU-DHL4 (DHL4), SU-DHL2 (DHL2), SU-DHL6 (DHL6), SU-DHL8 (DHL8), BJAB, WSU-NHL (WSU), Farage, U2932, K422, Toledo, TMD8, HBL1, OCI-Ly1 (Ly1), OCI-Ly3 (Ly3), OCI-Ly4 (Ly4), OCI-Ly7 (Ly7), OCI-Ly10 (Ly10), OCI-Ly18 (Ly18), OCI-Ly19 (Ly19) were grown as described in Supplementary Materials and Methods. Frozen primary DLBCL cells were obtained from the Biobank at the Department of Pathology, Uppsala University Hospital following ethical approval by the local ethical review board (approval number Dnr2014/ 233) and were purified by negative selection using CD3, CD14, and CD16 antibodies (>97% CD19 + cells after purification).
Analysis of synergy following treatment with venetoclax + R406 or venetoclax + ibrutinib was done by determining the combination index (CI) using the method of Chou and Talalay [27] and the software package CompuSyn (ComboSyn Inc., Paramus, NJ). The effects of MCL1 downregulation and BIM, HRK, or SHP1 overexpression on venetoclax sensitivity were evaluated by comparing observed vs. expected tumor cell survival, as described elsewhere [28, 29]. Other statistical analyses, as well as transfection, immunoblotting, RQ-PCR, and in vivo xenografting experiments are described in Supplementary Materials and Methods.

Results

R406 synergistically enhances the cytotoxic activity of venetoclax in BCR-dependent DLBCL cell lines

To understand the mechanisms that determine venetoclax sensitivity in DLBCL, we first tested the cytotoxic effect of venetoclax by Annexin V/PI staining against a panel of 18 DLBCL cell lines (12 GCB and 6 ABC) (Supplementary Table 1). Five of the GCB and four of the ABC DLBCL cell lines had previously been classified as BCR-dependent by comprehensive consensus clustering (CCC) analysis, whereas the remaining cell lines were classified as non-BCR or had not been classified using this approach [18, 20, 30]. Ten of the investigated cell lines (7 GCB and 3 ABC) expressed high levels of BCL-2, whereas BCL-2 was undetectable or expressed at low levels in the remaining 8 cell lines (Supplementary Table 1 and Supplementary Fig. 1). At concentrations ranging up to 0.25 μM, venetoclax had only modest activity against most BCL-2-positive DLBCL cell lines and was completely or almost completely ineffective against any of the BCL-2-low/negative cell lines (Fig. 1a and Supplementary Table 1). However, pretreatment with the SYK inhibitor R406 at clinically achievable concentrations for 2 h markedly increased the cytotoxic activity of venetoclax, resulting in a strong synergistic effect in 7 and a more modest synergistic effect in 3 DLBCL cell lines (Fig. 1b and Supplementary Fig. 2). This synergistic effect was seen with both GCB (n= 6) and ABC (n= 4) DLBCL cell lines and occurred in the absence of a substantial cytotoxic effect of R406 on its own (>75% viability with 2 μM R406 in all cell lines except WSUNHL). Importantly, seven of the synergistically sensitive cell lines had previously been classified as BCR-dependent and 9 of them expressed high levels of BCL-2, suggesting that BCR activation and BCL-2 overexpression determine sensitivity to the venetoclax + R406 combination.
To understand the mechanism(s) through which R406 increases the sensitivity of DLBCL cells to venetoclax, we evaluated changes in the expression of several BCL-2 family proteins that have been implicated in venetoclax resistance, including the antiapoptotic proteins MCL-1, BCL-xL, BCL-2, and A1 and the proapoptotic proteins BIM and HRK. The levels of MCL-1, BIM, BCL-2, BCLxL, and A1 were evaluated by immunoblotting analysis, whereas HRK was analyzed by RQ-PCR because of the unavailability of a specific antibody.
Changes in expression were evaluated after 24 h treatment with 2 μM R406 (Fig. 2 and Supplementary Fig. 3). A significant change was observed in the levels of MCL-1, which was downregulated in 8 of the 10 synergistically sensitive and only 1 of the 8 non-synergistically sensitive cell lines (P= 0.015 with the Fisher Exact Test) (Fig. 2). In addition, BIM was upregulated in 7 and HRK in 5 of the synergistically sensitive DLBCL cell lines, whereas no significant changes were observed in the levels of BCL-2, BCL-xL, and A1.

Ibrutinib displays similar effects as R406 in BCRdependent cell lines

To confirm that the observed effects of R406 are a consequence of targeting the BCR pathway, we repeated the above described experiments using the BTK inhibitor ibrutinib. When used at the same concentration as R406 (0.5–2.0 μM), ibrutinib displayed synergistic activity against 8 of the 10 venetoclax + R406 sensitive cell lines (4 GCB and 4 ABC) (Fig. 3a). Synergistic activity was also observed using lower, clinically achievable concentrations of ibrutinib (0.125 and 0.25 μM) and was confirmed in an independent set of experiments by analysis of caspase 3 activation (Supplementary Fig. 4). Moreover, ibrutinib induced similar changes as R406 in the expression of MCL1 and BIM in the 5 investigated DLBCL cell lines (3 GCB and 2 ABC) and in 3 of the 5 investigated patient-derived tumor cell samples (Fig. 3b). These effects were observed in both GCB (DHL4, Ly1 and DHL6) and ABC DLBCL cell lines (HBL1 and U2932) and were seen using both high (1 or 2 μM) and low (0.125 and 0.25 μM) ibrutinib concentrations (Fig. 3b and Supplementary Fig. 5, respectively). In contrast, the ibrutinib-induced changes in HRK expression were seen only at high concentrations, suggesting an off-target effect of ibrutinib (Supplementary Fig. 5). Consistent with this possibility, we observed that downregulation of BTK by RNA interference induces changes only in the levels of MCL-1 and BIM, while having no effect on the levels of HRK (Fig. 3c). In contrast, downregulation of SYK mirrored the previously observed effects with R406 on MCL-1, BIM, and HRK expression. These effects were not due to changes in cell viability, as no differences were observed in the ratio of cleaved vs. intact PARP or in the percentage of Annexin V/PI positive cells after treatment with control vs. SYK or BTK siRNA (Fig. 3d and Supplementary Fig. 6). Together, these data suggest that MCL1 and BIM are regulated through a common pathway downstream of SYK and BTK, whereas HRK is regulated downstream of SYK through a BTKindependent pathway.

R406 and ibrutinib inhibit PI3K/AKT and ERK signaling in BCR-dependent DLBCL cell lines

To characterize the mechanisms through which R406 and ibrutinib affect the expression of MCL-1 and BIM, we investigated the effects of these two drugs on the activity of several downstream BCR signaling molecules. These included AKT, FOXO, and ERK, which have been reported to regulate BIM transcription and turnover [31–33], and GSK3 and mTORC1, which have been reported to regulate MCL-1 turnover and translation [34, 35]. The effects of ibrutinib and R406 were investigated using phosphospecific antibodies that recognize the activated forms of AKT, ERK, and S6 ribosomal protein (analyzed as an indicator of mTORC1 activity) and the inactivated forms of GSK3 and FOXO. As shown in Fig. 4a, both R406 (2 μM) and ibrutinib (0.25 and 2.0 μM) reduced the levels of phospho-AKT, phosphoFOXO, and phospho-GSK3 in each of the 4 investigated BCR-dependent cell lines. In addition, a reduction in phospho-ERK was observed in DHL6 and HBL1, whereas a modest reduction in phospho-S6 was observed in Ly1. Of note, R406 was generally more effective than ibrutinib in reducing the levels of phospho-AKT, phospho-FOXO, and phospho-GSK3, whereas no difference was observed with respect to phospho-ERK. The levels of phospho-AKT, phospho-FOXO, and phospho-GSK3 also decreased following siRNA-mediated knockdown of SYK or BTK in DHL6 cells, although in this case no difference was observed between the two conditions, presumably because of the more efficient downregulation of BTK (Fig. 4b). Collectively, these data suggest that both drugs can inhibit the PI3K/AKT and ERK pathways, but the effects of R406 on the PI3K/ AKT pathway appear to be greater, consistent with our previous observations in CLL B cells [13].

Changes in MCL-1, BIM, and HRK expression sensitize DLBCL cells to venetoclax

To investigate how the observed changes in MCL-1, BIM, and HRK influence sensitivity to venetoclax, we transfected DHL4, HBL1, U2932, and LY1 cells with MCL-1 siRNA or BIM (BIMEL+ BIML) or HRK mRNA and evaluated cell viability in the presence or absence of venetoclax (Fig. 5). Downregulation of MCL-1 and overexpression of BIM or HRK resulted in variable induction of apoptosis in the different cell lines, ranging from relatively modest effects to massive cell killing. Co-treatment with venetoclax significantly increased the percentage of apoptotic cells both with respect to non-treated cells, as well as with respect to venetoclax-treated cells transfected with control siRNA or control mRNA (Fig. 5b). Comparison of observed vs. expected tumor cell survival indicated a prominent synergistic sensitizing effect in cells with downregulated MCL-1, whereas overexpression of BIM and HRK had mainly additive or weakly synergistic effects (Fig. 5c). Together, these data suggest that the venetoclax-sensitizing effects of R406 and ibrutinib are primarily mediated by downregulation of MCL-1.

R406 and ibrutinib synergize with venetoclax in an in vivo GCB DLBCL model

To further assess the capacity of R406 and ibrutinib to synergize with venetoclax, we tested the activity of these drugs alone or in combination in immunodeficient NSG mice xenografted with the GCB DLBCL cell line DHL6. Tumors were injected subcutaneously in the right flank and allowed to grow until they reached a size of >250 mm3 before initiating treatment. Treatment was administered by intraperitoneal injections using previously reported doses and schedules for fostamatinib, ibrutinib, and venetoclax [36–38]. Each drug alone partially inhibited tumor growth, but the combinations displayed significantly greater activity and in some cases induced tumor regression (Fig. 6). Significant differences in tumor volume between mice receiving combination treatment and mice receiving vehicle control or single agent treatment were detected starting from day 11 and remained significant until day 18, when treatment was discontinued. No difference was observed between mice receiving venetoclax in combination with fostamatinib or ibrutinib, confirming that both drugs can enhance the activity of venetoclax in this GCB DLBCL model.

SHP1 deficiency is responsible for the constitutive activation of the BCR pathway in GCB DLBCL

As previously mentioned, expression of autoreactive BCRs and the presence of gain-of-function mutations in the CD79A or CD79B subunit have been shown to be responsible for the chronic activation of the BCR pathway in ABC DLBCL [19, 21]. In GCB DLBCL the mechanism responsible for activation of the BCR pathway is still unknown, but recent studies have reported that a substantial proportion of DLBCL cases lack expression of the phosphatase SHP1 (PTPN6) [39, 40]. Because SHP1 is a key negative regulator of the BCR pathway, we correlated lack of SHP1 with R406 sensitivity. Remarkably, all of the GCB DLBCL cell lines (Ly1, Ly4, Ly18, DHL4, DHL6 and WSU-NHL) and 2 of the 4 ABC DLBCL cell lines (U2932 and HBL1) that were synergistically sensitive to the venetoclax + R406 combination did not express or expressed markedly reduced levels of SHP1 (Fig. 7a). Reduced or absent expression of SHP1 was associated with the presence of phosphorylated SYK (pSYK-Y352) and BLNK (pBLNK-Y84), suggesting that these two BCR-proximal signaling molecules are activated because of SHP1 deficiency. To further explore this possibility, we re-expressed SHP1 in Ly1 and DHL4 cells by transient transfection with a plasmid expression vector and investigated the effect on SYK and BLNK phosphorylation (Fig. 7b). A substantial reduction in the levels of phosphorylated SYK and phosphorylated BLNK was observed in both cell lines, which was confirmed in a separate set of experiments by lentiviral transduction of SHP1 (Fig. 7c). Expression of SHP1 also resulted in reduced levels of phosphorylated (inactivated) GSK3 (Fig. 7b) [13]. Importantly, these effects were accompanied with increased sensitivity to venetoclaxmediated cell killing (Fig. 7d), as well as changes in the expression of MCL1, BIM, and HRK that mirrored those induced by inhibition or downregulation of SYK (Fig. 7e).
To determine whether SHP1 is selectively downregulated in DLBCL cells compared to normal germinal center B cells, we compared the expression profiles of various normal human B cell subsets and primary B cell tumors from a publicly available gene expression dataset (Supplementary Fig. 7). In contrast to normal centrocytes and centroblasts, which showed rather uniform SHP1 expression, approximately 50% of DLBCL tumors showed downregulation of SHP1. Analysis of 3 additional datasets comprising more than 1000 DLBCL samples showed variable expression of SHP-1 and significantly more frequent downregulation in GCB compared to ABC DLBCL tumors. Collectively, these data suggest that loss of SHP1 expression represents a frequent mechanism of BCR pathway activation in GCB DLBCL.

Discussion

The BCR pathway plays a prominent role in the pathogenesis of various B cell malignancies and has recently emerged as a major therapeutic target in several of these diseases. Previous studies by us and others have shown that this pathway is particularly important in CLL, where it protects the malignant B cells from spontaneous and therapy-induced apoptosis, including apoptosis induced by novel agents such as venetoclax [13, 15, 16]. We now show that BCR signals are also involved in mediating venetoclax resistance in a substantial proportion of DLBCL cases and identify a novel mechanism of BCR pathway activation that appears to be particularly relevant for the GCB subtype of DLBCL. As such, this study provides further rationale for combining venetoclax with BCR signaling inhibitors in DLBCL and identifies a potential novel predictive biomarker for patients that are more likely to respond to this combination.
Previous in vitro studies have shown that prolonged treatment with the SYK inhibitors R406 or PRT060318 can inhibit the proliferation and induce apoptosis in BCR-dependent DLBCL cell lines belonging to both cell-of-origin subtypes [18, 41]. Prolonged treatment with ibrutinib has also been reported to inhibit proliferation and induce apoptosis in BCR-dependent ABC DLBCL cell lines [19], but the effects of this drug against BCR-dependent GCB DLBCL cell lines had not been investigated before. In a recent phase 1/2 clinical trial ibrutinib displayed greater activity in ABC than GCB DLBCL, with 37% of patients with ABC and 5% of patients with GCB DLBCL responding to treatment, respectively [22]. Fostamatinib, on the other hand, demonstrated lower activity than ibrutinib in 2 separate trials of relapsed/refractory DLBCL, but no preference for a particular DLBCL subset was observed [25, 26]. The reason for this different activity is still not understood, but could possibly reflect a greater dependency of ABC vs. GCB DLBCL cells on BCR signals and/or a greater capacity of ibrutinib to target the NF-kB pathway, which is selectively activated in the ABC DLBCL subtype.
Despite the different activity of fostamatinib and ibrutinib in these clinical trials, we observed that both drugs can sensitize DLBCL cell lines to venetoclax, independently of the cell-of-origin subtype. This sensitizing effect occurred after a relatively short drug exposure, insufficient to induce cell killing on its own, and was associated in most cases with downregulation of MCL-1 and upregulation of BIM. Both of these proteins have previously been reported to be regulated by the PI3K/AKT pathway, which is activated downstream of the BCR in both cell-of-origin subtypes [20, 23, 24, 42]. Importantly, we observed that exposure to R406 or ibrutinib, as well as downregulation of SYK or BTK inhibits the phosphorylation of AKT and its immediate substrates GSK3 and FOXO, suggesting that changes in the activity of these proteins mediate the effects on MCL1 and BIM. Together, these findings suggest that inhibition of the PI3K/AKT pathway is responsible for the synergizing effects of R406 and ibrutinib and provide an explanation why cell lines from both DLBCL subtypes are sensitized by these drugs despite the preferential activity of single-agent ibrutinib in ABC DLBCL.
Although both drugs displayed synergistic activity in the in vitro and in vivo experiments, it is worth noting that ibrutinib was less effective than R406 in inhibiting AKT, GSK3, and FOXO phosphorylation even when used at concentrations that exceed those achievable in patients. This finding is not surprising, considering that BTK is activated in parallel to AKT and that BTK deficiency has been reported to only partially reduce AKT activation through an apparently indirect mechanism [43, 44]. Moreover, these findings are consistent with our recent findings in CLL, which showed less efficient downregulation of MCL-1 in BCR-stimulated CLL cells by ibrutinib compared to R406 or entospletinib [13], which is another SYK inhibitor currently in clinical development. Together, these data suggest that SYK inhibitors could be more effective than ibrutinib in sensitizing malignant B cells with an activated BCR pathway to venetoclax. However, considering the different pharmacokinetics and the superior single agent activity of ibrutinib in ABC DLBCL, it becomes impossible to currently predict which drug combination would be more effective in the clinical setting.
The second main novel finding of this study is the observation that GCB DLBCL cell lines previously defined as BCR-dependent uniformly lack SHP-1 expression. This phosphatase is the principal negative regulator of the BCR pathway and functions by dephosphorylating several BCRproximal signaling molecules, including CD79A, CD79B, SYK, and BLNK [45, 46]. The presence of phosphorylated SYK and BLNK in BCR-dependent GCB DLBCL cell lines has been reported before [18, 47] and was found to strongly correlate with SHP-1 deficiency in our current study. Moreover, re-expression of SHP1 in these cell lines was shown to result in reduced levels of phosphorylated SYK and BLNK, further suggesting that loss of SHP1 is responsible for the constitutive activation of the BCR pathway in GCB DLBCL cells. In further support of this possibility, we observed that re-expression of SHP-1 in GCB DLBCL cell lines replicates the changes in MCL1, BIM, and HRK expression observed following inhibition or downregulation of SYK and sensitizes these cells to venetoclax. Collectively, these findings suggest that at least two different mechanisms may account for activation of the BCR pathway in DLBCL: an antigen-dependent mechanism resulting in a “chronic active” BCR signal that characterizes the ABC DLBCL subset and an antigen-independent mechanism resulting in an exaggerated “tonic” BCR signal that characterizes the GCB DLBCL subset. The different quality of the signals generated by these 2 mechanisms may explain the difference in NF-kB activity between the 2 DLBCL subsets.
Previous immunohistochemical studies have reported that SHP1 is downregulated in a number of B cell malignancies characterized by constitutive or excessive BCR pathway activation, including DLBCL, Burkitt lymphoma, follicular lymphoma, and mantle cell lymphoma [39, 40, 48–51]. These findings were corroborated in the current study through the analysis of a public gene expression dataset showing that SHP1 mRNA levels are reduced in a substantial proportion of DLBCL, Burkitt and follicular lymphoma tumors in comparison with normal germinal center B cells. In Burkitt lymphoma, SHP1 has been reported to be repressed because of activating mutations in the transcription factor TCF3 or inactivating mutations in its negative regulator ID3 [51]. Such mutations are rare in DLBCL [52], but mutations in SHP1 itself were recently identified in approximately 5% of primary tumors in two large studies investigating the genetic profile of DLBCL [53, 54]. In addition, experiments with murine GC B cells have shown that genetic ablation of the histone methyltransferase KMT2D, which is inactivated by various genetic alterations in approximately 30% of DLBCL tumors, results in downregulation of SHP1 [55], whereas experiments with human DHL4 cells have shown that knockdown of the transcriptional repressor BCL6, which is commonly overexpressed in DLBCL, induces SHP1 expression [56]. Together, these data suggest that downregulation of SHP-1 may represent a common mechanism of BCR pathway activation in GCB DLBCL, Burkitt lymphoma and perhaps other B cell malignancies, although the genetic defects responsible for SHP-1 downregulation are apparently different.
In addition to SHP1, repression of the phosphatase PTPROt has been implicated as a potential mechanism for enhanced tonic BCR signaling in GCB DLBCL. In an earlier study, Aguiar et al. reported that this phosphatase, which is abundantly expressed in normal naive B cells, is decreased or absent in normal GC B cells and GC-derived DLBCLs [57]. Although the role of this phosphatase as a negative regulator of the BCR pathway has not been as firmly established as in the case of SHP1, overexpression experiments in DHL-4 cells showed that it can also inhibit BCR-triggered SYK and BLNK phosphorylation [58]. Further studies will be required to determine the relationship between loss of SHP1 and loss of PTPROt expression and the relative contribution of these two events for the constitutive activation of the BCR pathway in GCB DLBCL.
While this manuscript was undergoing review, a paper was published reporting that the PI3Kα/δ inhibitor copanlisib can also synergize with venetoclax in BCR-dependent DLBCLs belonging to both cell-of-origin subtypes [59]. The results of this study are in agreement with our findings and provide further rationale for clinical testing of combinations of venetoclax and BCR inhibitors in selected DLBCL patients. Moreover, this study provides additional evidence that the PI3K/AKT pathway mediates venetoclax resistance in BCL2-positive BCR-dependent DLBCL.

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