SYK is activated by mutated MYD88 and drives pro-survival signaling in MYD88 driven B-cell lymphomas

Activating MYD88 mutations promote pro-survival signaling through BTK and HCK, both targets of ibrutinib. Despite high response rates, complete responses to ibrutinib are lacking, and other MYD88 triggered pro-survival pathways may contribute to primary drug resistance. B-cell receptor (BCR) signaling has been observed in lymphomas driven by mutated MYD88, even without activating the BCR pathway mutations. We identified activated SYK (p-SYK), a component of BCR in complex with MYD88 in MYD88-mutated WM and ABC DLBCL lymphoma cells. Confocal microscopy confirmed co-localization of MYD88 with SYK in MYD88-mutated cells. Knockdown of MYD88 or use of a MYD88 signaling inhibitor abrogated SYK activation, while expression of mutated but not wild-type MYD88 amplified p-SYK in MYD88-mutated and wild-type lymphoma cells. Knockdown of SYK or use of inhibitors targeting SYK blocked p-STAT3 and p-AKT signaling in MYD88-mutated cells. Cell viability analysis showed that combining ibrutinib and SYK inhibitors triggered synthetic killing of MYD88-mutated lymphoma cells. Our findings extend the spectrum of mutated MYD88 pro-survival signaling to include SYK directed BCR cross talk in MYD88-mutated lymphomas. Targeting SYK in combination with ibrutinib produces synthetic lethality, providing a framework for the clinical investigation of ibrutinib with SYK inhibitors in MYD88-mutated lymphomas.

Activation of B-cell receptor (BCR) signaling is common among MYD88-mutated B-cell malignancies. Activating CD79A/B mutations are frequently present in ABC DLBCL and PCNSL and can trigger SYK-mediated downstream signaling 2,11,12 . In contrast to these aggressive entities, CD79A/B activating mutations are uncommon in WM with a reported incidence of 3-12%, and have been associated with transformed WM disease [13][14][15][16] . Despite the absence of activating BCR mutations, active B-cell receptor (BCR) signaling has been observed in WM and ABC DLBCL patients, and could contribute to prosurvival signaling [17][18][19] . We therefore have sought to clarify whether mutated MYD88 could account for chronic BCR signaling in MYD88-mutated lymphoma cells. While BTK, a component of the BCR signalosome is triggered by mutated MYD88, our previous work showed that the upstream BCR signaling member SYK was activated in MYD88-mutated WM patient cells 17 . Moreover, the SYK inhibitor fostamatinib triggered apoptosis of MYD88-mutated WM cells, suggesting an important function for SYK in MYD88 pro-survival signaling 20 . We therefore investigated a role for SYK as a mediator of BCR activation in MYD88-mutated lymphomas. Knowledge of MYD88-directed SYK signaling could prompt development of novel therapeutic approaches aimed at extinguishing other routes for MYD88-directed pro-survival signaling beyond ibrutinib and other BTK inhibitors.

Patient samples and treatments
Mononuclear cells from freshly obtained WM patient's bone marrow (BM) aspirates were isolated using Ficoll-Paque™ PLUS Media (GE Healthcare™) and treated with either ibrutinib, SYK inhibitor tamatinib or entospletinib, or combination of ibrutinib and a SYK inhibitor. Apoptosis analyses were performed on MYD88 genotyped CD19-gated lymphoplasmacytic cells (LPCs) following overnight treatment of BM mononuclear cells, as previously described 5,6,23 . Subject participation was approved by the Harvard Cancer Center/Dana-Farber Cancer Institute Institutional Review Board, and all participants provided written consent for sample use.

Co-immunoprecipitation experiments
Co-immunoprecipitation studies were performed as previously described 5 using anti-MYD88 antibody (Santa Cruz Biotechnology) and SYK, p-SYK(Y525/Y526) antibodies (Cell Signaling Technologies). Briefly, cells were lysed with Co-IP buffer (Thermo Fisher Scientific) supplemented with 1 mM sodium orthovanadate, 10 mM NaF, 1 × protease inhibitors cocktail for 15 min on ice, and then centrifuged at 2600 × g for 5 min. Supernatants (2 mg total protein) were incubated with 2-4 µg of antibodies at 4°C for 30 min, followed by incubation with protein A/Gcoated magnetic beads (EMD Millipore) for another 30 min at 4°C. After samples were washed four times with ice-cold lysis buffer on a magnetic stand, proteins were eluted using SDS-PAGE loading buffer for further analysis.

Cytotoxicity studies
The CellTiter-Glo® Luminescent cell viability assay (Promega, Madison, WI) was used to assess the dose response of inhibitors alone or in combination 5,6 . Cells were seeded into 384 or 96-well plates with the EL406 Combination Washer Dispenser (BioTek Instruments, Inc.), and inhibitors were injected into culture media with the JANUS Automated Workstation (PerkinElmer Inc., Waltham, MA, USA). Cells were incubated with inhibitors for 72 h at 37°C. Luminescent measurements to assess cell viability were performed using the 2104 Envision® Multilabel Reader (PerkinElmer Inc.). Drug interactions were assessed by CalcuSyn 2.0 software (Biosoft, Cambridge, UK) based on Chou TC 24 .

Reproducibility and statistical analysis
Sample size of experiments and the number of experiments performed are depicted in the figure legends. The statistical significance of differences was analyzed using one-way ANOVA with Tukey's multiple comparisons test by Prism software. Differences were considered significant when p < 0.05. Error bars denote standard deviation.

SYK is activated in MYD88-mutated lymphoma cells, and its activation is regulated by MYD88
A cross cell line comparison of the phosphorylation levels of SYK (pY525-pY526) showed higher levels of activated SYK in MYD88-mutated versus wild-type cell lines, while ABC DLBCL cells (TMD8, HBL-1) with both MYD88 and CD79B mutations expressed higher levels of phosphorylated SYK versus MYD88 only mutated WM cells (Fig. 1a). The highest p-SYK levels were observed in OCI-Ly3 ABC DLBCL cells that are homozygous for MYD88 L265P (Fig. 1a). More pronounced SYK phosphorylation was also identified in primary MYD88mutated WM cells when compared with healthy donor peripheral B cells (Fig. 1b). We next performed Phos-Flow studies for SYK in MYD88-mutated lymphoma cells following treatment with a MYD88 inhibitor or control peptide. Akin to our previous findings for NFkB signaling 1,5 , these studies also showed that phosphorylation of SYK at Tyrosine 525 and 526 (pY525-pY526) was reduced following treatment with a MYD88 peptide inhibitor in both MYD88-mutated WM and ABC DLBCL cells (Fig. 1c). Noteworthy, SYK phosphorylation was more robustly reduced in the WM cell lines (BCWM.1, MWCL-1), as well as the ABC DLBCL cell line OCI-Ly3 that carry only the MYD88 L265P mutation, but negligible in ABC DLBCL cells (TMD8, HBL-1) with both MYD88 and CD79B activating mutations (Fig. 1c). Treatment of primary MYD88-mutated WM cells with the MYD88 peptide inhibitor also blocked SYK phosphorylation (Fig. 1d).
To further explore the possibility that SYK was activated by mutated MYD88, we performed a knockdown of MYD88 in MYD88-mutated BCWM.1 WM cells and MYD88/CD79B mutated TMD8 ABC DLBCL cells. While these experiments showed a marked reduction in phosphorylation of SYK (pY525-pY526), and the SYK downstream components STAT3 (pY705) and AKT (pS473) in BCWM.1, these changes were not observed in TMD8 cells, consistent with our prior findings using MYD88 inhibitor peptides (Fig. 2a). As shown in our prior studies, p-BTK (Y223) expression was decreased by MYD88 knockdown in both BCWM.1 and TMD8 cells 5 . Importantly, the expression of MYD88 L265P but not wild-type MYD88 increased phosphorylated SYK in MYD88mutated BCWM.1, as well as in MYD88 wild-type OCI-Ly7 and Ramos cell lines (Fig. 2b).
Activated SYK is a component of the "Myddosome" signaling complex in MYD88-mutated lymphoma cells Since SYK was activated by mutated MYD88, we next sought to clarify if SYK and p-SYK were present in the "Myddosome" signaling complex in MYD88-mutated lymphoma cells. We therefore performed coimmunoprecipitation (Co-IP) experiments using a MYD88-binding antibody, as well as reverse Co-IP experiments using both SYK and p-SYK antibodies in MYD88-mutated BCWM.1 and MYD88 wild-type Ramos cells. The MYD88 antibody effectively pulled down SYK in Co-IP experiments, and both SYK and p-SYK antibodies pulled down MYD88, with more robust pulldown of MYD88 by activated SYK in MYD88mutated BCWM.1 cells (Fig. 3a). Conversely, both SYK and p-SYK antibodies failed to pull down MYD88 in MYD88 wild-type Ramos cells (Fig. 3a). As expected, the IP of p-SYK did not reveal SYK itself in line with the results shown in Fig. 1a that showed lack of p-SYK in MYD88 wild-type cells. Further to these findings, we performed cellular co-localization experiments using immunofluorescent (IF) with antibodies for MYD88 and SYK in both MYD88-mutated and wild-type lymphoma cells. By confocal microscopy, IF staining showed that MYD88 and SYK co-localized in the cytoplasm with a punctate staining pattern in MYD88-mutated BCWM.1, MWCL-1 and TMD8 cells (Fig. 3b), while the MYD88 showed less punctate staining pattern and not co-localized with SYK in MYD88 wild-type Ramos, OCI-Ly7, and OCI-Ly19 cells (Fig. 3b)  The results from PhosFlow studies for p-SYK (pY525-pY526) following treatment with a MYD88 inhibitor or control peptide in lymphoma cells carrying only mutated MYD88, or mutated MYD88 and CD79B. Representative histograms and relative median fluorescence intensity from three independent experiments are shown (c). Findings from PhosFlow studies for p-SYK (pY525-pY526) following treatment of primary MYD88-mutated WM cells from two patients with a MYD88 inhibitor or control peptide (d).

Activated SYK promotes STAT3 and AKT signaling as an intermediary of mutated MYD88
To explore the downstream events of activated SYK in MYD88-mutated WM cells, we next knocked down SYK using two lentiviral vectors in BCWM.1 cells. Knockdown of SYK reduced the phosphorylation of STAT3 (pY705) and AKT (pS473), while only modest or no changes in BTK, IRAK1 and IRAK4 phosphorylation were observed (Fig. 4a). In addition to these experiments, we treated MYD88-mutated WM and ABC DLBCL cells with tamatinib or entospletinib, and examined changes in the phosphorylation levels of SYK, STAT3, and AKT. As expected, tamatinib as well as entospletinib effectively blocked the phosphorylation of SYK, and its downstream components STAT3 and AKT in MYD88 only mutated WM cells, as well as in ABC DLBCL cell lines carrying both MYD88 and CD79B activating mutations (Fig. 4b).
To further clarify the downstream signaling pathways impacted by SYK in MYD88-mutated WM cells, we engineered BCWM.1 cells to express vector only, wildtype MYD88 or MYD88 L265P and treated them with the SYK inhibitors tamatinib or entospletinib. The increased phosphorylation of SYK (pY525-pY526), as well as the known downstream signaling components STAT3 (pY705) and AKT (pS473) induced by the expression of MYD88 L265P were blocked by both SYK inhibitors in a dose-dependent manner (Fig. 4c).

SYK supports growth and survival of MYD88-mutated lymphoma cells
Since SYK activation is directly caused by mutated MYD88 and mediates its downstream STAT3 and AKT signaling, we next sought to clarify the importance of SYK in supporting cell growth and survival in MYD88-mutated  lymphoma cells. We therefore assessed the cell growth and survival of MYD88-mutated WM and ABC DLBCL cells, including MYD88/CD79B mutated TMD8 and HBL-1 cells using CellTiter-Glo® Luminescent cell viability assay following SYK knockdown by lentiviral transduction. GFP-positive cells were sorted on day 5 following lentiviral transduction, and the cell growth and survival were determined every 2 days until day 11. Compared with scrambled control, knockdown of SYK reduced cell growth and survival in BCWM.1, MWCL-1, and TMD8 cells (Fig. 5a). Moreover, treatment of MYD88-mutated WM and MYD88/CD79B ABC DLBCL cell lines with tamatinib or entospletinib showed killing at pharmacologically achievable levels for either agent (Fig. 5b) 21,25 .
The combined inhibition of BTK and SYK produces synergistic lethality in MYD88-mutated lymphoma cells Since SYK provides a divergent stream of pro-survival signaling from previously identified mutated MYD88 driven BTK and HCK, both targets of ibrutinib, we next sought to examine the combined effects of ibrutinib and (see figure on previous page) Fig. 3 Activated SYK is a component of the "Myddosome" signaling complex in MYD88-mutated lymphoma cells. Western blots showing SYK pulldown by MYD88 antibody and MYD88 pulldowns by SYK and p-SYK antibodies in MYD88-mutated and wild-type cells in co-IP experiments. Experiments were run using 2 mg of total protein in duplicated samples, and IgG levels used to demonstrate uniform antibody addition for pulldown experiments (a). Confocal microscopy images showing nuclear staining by DAPI (blue); MYD88 staining by anti-MYD88-Alexa Fluor® 488 (green); and SYK staining by anti-SYK-Alexa Fluor® 647 (red) antibodies. MYD88 and SYK protein co-localization appears in the cytoplasm (yellow) with a punctate staining pattern following merging of MYD88 and SYK staining images in MYD88-mutated BCWM.1 and MWCL-1 WM cells, and TMD8 ABC DLBCL cells, but not in MYD88 wild-type Ramos Burkitt's lymphoma cells, or OCI-Ly7 and OCI-Ly19 GCB DLBCL cells (b). Studies were performed twice, and representative experimental set is shown. either tamatinib or entospletinib. The combination of ibrutinib with either SYK inhibitor produced increased lethality in MYD88-mutated BCWM.1 and MWCL-1 WM cells, as well as MYD88/CD79B mutated TMD8 and HBL-1 ABC DLBCL cells. Combination index (CI) and normalized isobologram analysis indicated ibrutinib and SYK inhibitor interactions were synergistic across nearly all doses that were evaluated (Fig. 6a-d).

Discussion
Despite the rarity of BCR pathway mutations in WM and their absence in many patients with ABC DLBCL, there are indications that chronic active BCR signaling is occurring in these entities. In previous studies, we identified that BTK, a downstream contributor to BCR signaling, was activated by mutated MYD88 through HCK and was incorporated into the "Myddosome" signaling complex that supported NFκB pro-survival signaling 5,6 . These findings enabled the investigation and fast-track approval of ibrutinib, a pleiotropic kinase that targets both BTK and HCK for WM. However, the activation of BTK by mutated MYD88 did not provide an explanation for SYK activation, an upstream BCR member that is activated in MYD88-mutated WM patient cells 17 . Moreover, the finding that fostamatinib triggered apoptosis of MYD88-mutated WM cells, suggested an important function for SYK in MYD88 prosurvival signaling 20 . We therefore investigated a role for SYK as a mediator of TLR/BCR cross talk in MYD88mutated lymphomas.
We observed that SYK in its active form was incorporated into the "Myddosome" signaling complex by both co-immunoprecipitation and co-localization experiments. Furthermore, by knockdown studies as well as use of SYK inhibitors, we validated the importance of SYK as an essential pro-survival molecule in MYD88-mutated lymphoma cells. The findings are consistent with those recently reported by Phelan et al. 26 , who described a MYD88-TLR9-BCR(IGM) (MY-T-BCR) super complex in MYD88-mutated tumor cells. Importantly, we found that SYK was activated by mutated MYD88, and served as an intermediary to trigger pro-survival STAT3 and AKT, but not BTK signaling. The latter finding is consistent with our earlier work that showed BTK activation was dependent on mutated MYD88 5 . Our current observations depict SYK and BTK, classically viewed as hierarchal components of BCR signaling, as "Myddosome" recruits that serve independent roles in propagating MYD88/TLR pathway signaling. Taken together, the findings add to the complexity and reach of mutated MYD88 to drive multiple pro-survival pathways that lead to NFκB, AKT, ERK, and STAT3 activation (Fig. 7).
The identification of SYK as a pro-survival signaling intermediary for mutated MYD88 may also explain the lack of complete responses reported with ibrutinib in MYD88-mutated B-cell malignancies, since SYK unlike BTK or HCK is not a target of these agents [6][7][8][9][10][11] . Importantly, the combination of SYK inhibitors with ibrutinib yielded synergistic lethality in MYD88-mutated lymphoma cells. These findings provide support for clinical trials combining SYK inhibitors with ibrutinib for MYD88-mutated lymphomas. Such an approach may be particularly suited to patients with both MYD88 and CD79 activating mutations since SYK remained active despite MYD88 inhibition in MYD88/CD79 doublemutated ABC cell lines. SYK inhibitors would therefore block upstream SYK activation mediated by activating CD79 mutations in these double-mutated tumors. Several SYK inhibitors, including fostamatinib, entospletinib, TAK-659, and HMPL-23 are currently in development for hematological and oncological indications. Fostamatinib was recently approved for the treatment of chronic ITP and showed significant single agent activity in previously treated CLL 27 . Entospletinib, a more selective SYK inhibitor, has also shown notable activity in previously treated CLL, with good tolerance and is currently being evaluated in combination with obinutuzumab in CLL, SLL, and NHL (NCT 03010358) 22,28 . The SYK inhibitor TAK-659 has also shown activity and good tolerance in FL, and DLBCL, including transformed cases 29 . A study evaluating TAK-695 in combination with chemotherapy is currently underway in high-risk DLBCL (NCT 03742258). Responses to the SYK inhibitor HMPL-3 have also been observed across multiple B-cell lymphomas, including WM 30 .
In summary, we have identified activated SYK as an integral component of the mutated MYD88 signaling apparatus, and an intermediary for AKT and STAT3 prosurvival signaling. Combined use of ibrutinib and SYK inhibitors produces synergistic lethality in MYD88mutated B cells, including those with accompanying CD79 activating mutations. The findings provide a framework for the clinical investigation of ibrutinib with SYK inhibitors in MYD88-mutated lymphomas. Mutated MYD88 triggers assembly of the "Myddosome" that includes activated BTK and IRAK4/IRAK1 that transactivate NFκB. Mutated MYD88 also transcriptionally upregulates and transactivates through IL-6 the SRC family member HCK, that triggers activation of BTK itself, as well as AKT, and ERK. Mutated MYD88 also activates SYK, a component of the "Myddosome" that triggers STAT3 and AKT.