# First-in-class inhibitor of HSP110 blocks BCR activation through SYK phosphorylation in diffuse large B-cell lymphoma

Vincent Cabaud Gibouin, Manon Durand, Christophe Boudesco, François Hermetet, Kristyna Nozickova, Catherine Chassagne-Clement, Mayssa Abdelwahed, Pavel Klener, Carmen Garrido, Gaëtan Jego

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Vincent Cabaud Gibouin, Manon Durand, Christophe Boudesco, François Hermetet, Kristyna Nozickova, et al.. First-in-class inhibitor of HSP110 blocks BCR activation through SYK phosphorylation in diffuse large B-cell lymphoma. Leukemia, 2024, 38 (8), pp.1742-1750. 10.1038/s41375-024-02302-x. hal-05263754

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# First-in-class inhibitor of HSP110 blocks BCR activation through SYK phosphorylation in diffuse large B-cell lymphoma

Vincent Cabaud Gibouin ID <fnref n="1" />,², Manon Durand ID <fnref n="1" />,², Christophe Boudesco <fnref n="1" />,², François Hermetet ID <fnref n="1" />,², Kristyna Nozickova³, Catherine Chassagne-Clement⁴, Mayssa Abdelwahed <fnref n="1" />,², Pavel Klener³, Carmen Garrido <fnref n="1" />,²,⁵ and Gaëtan Jego ID <fnref n="1" />,²✉

© The Author(s), under exclusive licence to Springer Nature Limited 2024

Activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL) is driven by aberrant activation of the B-cell receptor (BCR) and the TLR/MyD88 signaling pathways. The heat-shock protein HSP110 is a candidate for their regulation as it stabilizes MyD88. However, its role in overall BCR signaling remains unknown. Here, we used first-in-class HSP110 inhibitors to address this question. HSP110 inhibitors decreased the survival of several ABC-DLBCL cell lines in vitro and in vivo, and reduced the phosphorylation of BCR signaling kinases, including BTK and SYK. We identified an interaction between HSP110 and SYK and demonstrated that HSP110 promotes SYK phosphorylation. Finally, the combination of the HSP110 inhibitor with the PI3K inhibitor copanlisib decreases SYK/BTK and AKT phosphorylation synergistically, leading to suppression of tumor growth in cell line xenografts and strong reduction in patient-derived xenografts. In conclusion, by regulating the BCR/TLR signaling pathway, HSP110 inhibitors are potential drug candidates for ABC-DLBCL patients.

Leukemia; https://doi.org/10.1038/s41375-024-02302-x

INTRODUCTION

Diffuse large B-cell lymphoma (DLBCL) is the most frequent lymphoid malignancy in adults. Among DLBCL, activated B-cell-like (ABC-) DLBCL patients have the least favorable clinical outcomes [1]. These outcomes are potentially linked to chronic active B-cell receptor (BCR) signaling, which is considered a major pathogenetic mechanism [2, 3]. Downstream from the BCR, these tumors rely mostly on the constitutively activated nuclear factor κB (NFκB) pathway to grow and resist cell death [2].

In connection with BCR, a cascade of kinases, such as spleen tyrosine kinase (SYK) and Bruton's tyrosine kinase (BTK), contributes to the initiation of the signal [4, 5]. However, several components of the BCR and toll-receptor (TLR) signaling machinery also show frequent activating mutations, contributing to aberrant signaling and subsequent lymphoma aggressiveness. Among these, the mutation of the TLR adapter MyD88 (MyD88<sup>L265P</sup>) and of the BCR-associated protein CD79b (CD79b<sup>Y196</sup>) [6–8] often occur together and can synergize to promote the NFκB signaling. Independently of the BCR-BTK pathway, mutations of CARD11, BCL10 and amplification in MALT1 contribute to the sustenance of a robust NFκB signaling by forming the CARD11–BCL10–MALT1 (CBM) complex [9–13].

In addition, there is a strong interaction between BCR components, MyD88 and TLR9, in some patients through a multiprotein complex (My-T-BCR), thus reenforcing this tumor cell

addiction towards the BCR-dependent NFκB pathway [14]. Aberrant activation of the phosphoinositide 3-kinase (PI3K)/AKT/mTOR pathway is also observed in a ABC-DLBCL samples and is mainly under the dependence of the PI3Kδ isoform [15–18].

Several targeted therapies grounded in the identification of these signaling pathways have been tested, but often demonstrating limited or patient-subgroups specific efficacy. For instance, the selective and irreversible BTK inhibitor ibrutinib has shown great efficacy, but mostly in patients with MyD88 and CD79b double mutation [14, 19]. Despite these encouraging results, responses to ibrutinib treatment are variable or incomplete, and most patients acquire drug resistance. Regarding the PI3K pathway, copanlisib (BAY 80-6846), a dual inhibitor of PI3Kδ and PI3Kα, has shown potent efficacy in preclinical models, especially when combined with ibrutinib [20]. These pre-clinical models support clinical evaluation of combinations targeting both the BTK and PI3K pathways in ABC-DLBCL.

To complement the existing drug portfolio, novel molecular targets would be a way to overcome the resistance to current treatments and to develop future tailored therapies. Several heat shock proteins (HSPs) are involved in the proximal signaling of the BCR in leukemia and lymphoma, and they are thus at the center of interest for the discovery of new targets [21]. HSPs are molecular chaperones overexpressed by tumor cells, and they have unique cytoprotective properties [22, 23]. Among them,

<footnote n="1">¹INSERM, UMR1231, Université de Bourgogne, 21078 Dijon, France. ²Equipe Labellisée « Ligue Nationale Contre le Cancer », Paris, France. ³Institute of Pathological Physiology, First Faculty of Medicine, Charles University Prague, Prague, Czech Republic. ⁴Service de Biopathologie, Centre Léon Bérard, Lyon, France. ⁵Centre Georges François Leclerc, 21000 Dijon, France. ✉email: gaetan.jego@u-bourgogne.fr</footnote>
HSP110 has major roles in colorectal cancer [24–27]. It also appears to be an important player in DLBCL survival as it correlates with the aggressiveness and proliferation index in patients [28]. HSP110 expression correlates with c-MYC and Bcl6 protein expression in tumor biopsies of non-Hodgkin lymphomas, and HSP110 knockdown leads to decreases in both

oncogenes [29]. Furthermore, HSP110 expression correlates with MyD88 in patient lymph node biopsies, and HSP110, by chaperoning MyD88 and MyD88<sup>L265P</sup>, enhances survival of ABC-DLBCL cell lines. Therefore, HSP110 appears to be a new therapeutic target involved in the regulation of NFκB pathway cell dependency. In this context, the recent identification of small

<figure><img src="image_3.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_8.png" /></figure>
**Fig. 1** HSP110-specific inhibitors reduce DLBCL growth in vitro and in vivo. **A** In vitro cell growth of TMD8, OCI-Ly3 and HBL-1 after 48 h and 96 h of treatment with 10 µM of inhibitor (iHSP110) #33, #52 or control #61 measured by CellTiter-Glo. Mean ± stedv of 3 experiments. **B** Survival of OCI-Ly10, HBL1, TMD8, and OCI-Ly3 measured by Annexin-V/7AAD staining after 48 h of treatment with increasing concentrations of iHSP110 #33 (left) or iHSP110 #52 (right) (n = 3). **C** Immunoblot analysis of PARP, cleaved PARP, cleaved Caspase 3, Bcl2 and Bcl-xL in TMD8, HBL1 and OCI-Ly3 after 48 h of treatment with 10 µM of iHSP110 #52. **B**-actin served as a loading control (n = 3). **D** In vivo iHSP110 treatment reduces tumor growth in mice. Tumor volume monitoring of TMD8 tumor bearing NSG mice treated with the iHSP110 (10 mg/kg or 20 mg/kg) or vehicle. Animals were treated (i.p.) every 2 days. Mean volume (± SD) is represented (n = 10 per group). **E** Tumor weight measured at day 20 after treatment with vehicle or with iHSP110 as in **D** (n = 10 per group). **F** Ex vivo images of representative xenograft tumors at day 20 after treatment with vehicle or with iHSP110 as in **E**. \*\*P < 0.01; \*\*\*P < 0.001; \*\*\*\*P < 0.0001.

chemical-specific inhibitors of HSP110 has opened the way to
new combinational drug testing [30].

Given the emerging role of HSP110 in DLBCL and the
importance of the BCR pathway in the pathogenetic mechanism
of the disease, here we used the first HSP110 inhibitors in DLBCL
preclinical models to explore how they affect the BCR pathway.
We show that the treatment of DLCBL cell lines alters the BCR/
MyD88/ NFkB pathway leading to reduced growth and survival of
tumor cells in vitro and in vivo. Studying HSP110-BCR pathway
interactions at the molecular level, our results unveil that HSP110
promotes SYK phosphorylation via direct interaction. Furthermore,
targeting the BCR pathway with a HSP110 inhibitor and the PI3K
pathway with copanlisib led to complete blockage of DLBCL
tumor growth in vivo.

METHODS

Primary tumors and cell lines

Available in supplementary Material and Methods.

Transfection and lentiviral infections

DLBCL cell lines were transfected with the AMAXA Nucleofector 2b device (Lonza, Basel, Switzerland) and the Nucleofector kit T (OCI-Ly3) or V (TMD8). siRNA control (MISSION® siRNA Universal Negative Control #1, Sigma-Aldrich) or hsp1 targeting siRNA (Silencer™ Select Pre-Designed siRNAs, Life technologies, Saint-Aubin, France) were used. AMAXA programs were P005 (TMD8) and G016 (OCI-Ly3). HEK293T transfections were performed using FuGENE HD reagent (Promega, Madison, WI). HSP110-GFP and MyD88 plasmids were previously described [31]. pWZL Neo Myr Flag SYK plasmid was a gift from William Hahn & Jean Zhao (Addgene plasmid #20646; http://n2t.net/addgene:20646; RRID:Addgene_20646). pMIG-TEL-SYK retroviral expression vector was described previously [32] and was a kind gift of Pr. H. Jumaa (Institute of Immunology, Ulm University Medical Center, Germany). OCI-Ly3 cell line was infected by the vector as previously described [31].

Western blots and co-immunoprecipitation

Available in supplementary Material and Methods

Cell proliferation and survival

Synergy of inhibitors on cell growth and survival was determined by the
Bliss synergy score [33]. Complementary informations are available
in supplementary Material and Methods.

Immunohistochemistry and Duolink® proximity ligation assay (PLA)

Cell lines were fixed using 4% PBS-PFA, permeabilized (100% chilled methanol), and blocked (0.1% TBS-Tween20 with 3% BSA). For PLA on cell lines, primary antibodies were HSP110 (sc74550, Santa Cruz), HSP70 (ADI-SPA-812, Enzo Life), and SYK (ab155187, Abcam). Duolink® experiments were performed following the manufacturer's instructions (Sigma-Aldrich) and mounted with ProLong Gold medium with DAPI (Fisher). For PLA on FFPE tissues, antigens were unmasked using a universal unmasking buffer (ab208572, Abcam). Primary antibodies were HSP110 (ab109624) and phosphotyrosine (ab9319) from Abcam, and SYK (80460) from Cell Signaling. For fluorescent DuoLink experiments (Sigma-Aldrich), tissues were incubated with Sudan Black B (Sigma-Aldrich) and with DAPI (Sigma, D9542). The slides were mounted using Fluoromount-G (InVitroGen).

Microscopy images were captured using an Axio Imager 2 (Carl Zeiss Microscopy GmbH) with an AxioCam MRm CCD camera (Carl Zeiss GmbH) and analyzed with the spot detector plugin of ICY software.

ABC-DLBCL cell-line xenograft and patient-derived xenografts (PDX) experiments

8-week-old female NSG mice (Jackson Laboratory, Bar Harbor, ME, USA) were used for all xenografts. They were subcutaneously injected with TMD8 cells or PDX cells (VFN-D1/KTC) [34]. Once the animal developed palpable tumors (mice without palpable tumor after 5 days were excluded), mice were randomized based on level of engraftment and treated intraperitoneally with HSP110 inhibitor #52 (iHSP110 52) at 10 mg/ kg/inj (Q2Dx8) or with the vehicle at the same frequency, and/or treated intravenously with copanlisib at 5 mg/kg (at least 6 animals/group). The size of the tumor in each animal was measured in the indicated days in the three perpendicular dimensions using a digital caliper. The animal experiments were approved by the institutional animal care and use committee of the University of Burgundy, the French Ministry of Higher Education and Research (APAFIS25224), the Higher Education section of the Ministry of Education, Youth and Sports of the Czech Republic.

In vitro phosphorylation assay

Available in supplementary Material and Methods.

RESULTS

HSP110 inhibition impacts B lymphoma cells survival and BCR signaling

To determine the impact of HSP110 inhibition on cell growth and survival, we treated several ABC-DLBCL cell lines with two HSP110-targeting compounds (inhibitors #33 and #52) that were screened from a library of foldamers and selected by us for their capacity to specifically inhibit HSP110. As a control, we used a compound that did bind to HSP110 but that did not affect the chaperone activity (#61) [30]. With compounds #33 and #52 only, we observed a decrease in cell growth in TMD8 and HBL1 at as early as 48 h, and at 96 h for OCI-Ly3 when treated with the inhibitor #52 at 10 µM (Fig. 1A). Also, both compounds decreased cell survival of OCY-Ly10, TMD8, HBL1 and OCI-Ly3, in a dose-dependent manner (Fig. 1B). Since compound #52 seemed more efficient on ABC-DLBCL cell lines, we chose to use this compound for the rest of the study, unless specified in the text, and it is designated henceforth as iHSP110. The apoptosis was confirmed by PARP and caspase-3 cleavage and by the decrease in the antiapoptotic proteins Bcl-xL and Bcl2, two major survival DLBCL proteins (Fig. 1C). To confirm the efficacy of the HSP110 inhibitor in vivo, we performed TMD8 xenografts in NSG mice. We observed a tumor growth inhibition of 53% at 10 mg/kg and of 68% at 20 mg/kg compared to the control treatment (Fig. 1D, F), and a lower tumor weight at day 20 (Fig. 1E), without in vivo toxicity (Supplementary Fig. 1A, B). Taken together, these data confirmed that HSP110 is a valuable target in ABC-DLBCL and that our HPS110 inhibitors are effective in vitro and in vivo.

We then investigated the effect of our HSP110 inhibitors in the NFkB pathway. We observed, in contrast to the control compound #61, that HSP110 inhibitors #33 and #52 reduced P-IkB and P-p65 in OCI-Ly3 and TMD8 (Fig. 2A, B respectively). HSP110 is also
<figure><img src="image_9.png" /><figcaption>**Fig. 2** HSP110-specific inhibitors reduce NFκB and BCR signaling pathways. Immunoblot analysis of HSP110, P-IκB, IκB A and P-p65 and p65 B in OCI-Ly3 and TMD8 after treatment with HSP110 inhibitors (iHSP110, 10 µM) or control during 24 and/or 48 h (n = 3). C Immunoprecipitation (IP) of MyD88 in HBL1 cells followed by immunoblot using anti-HSP110, anti-MyD88, anti-immunoglobulin heavy chains and anti-vinculin antibodies (loading control). A nonrelevant antibody was used as a control (IP control). Cells were treated or not with iHSP110 20 µM or control during 24 h (n = 3). D Immunoblot analysis of P-PLCg2, PLCg2, P-BTK, BTK, P-SYK, SYK, in HBL1, OCI-Ly3 and TMD8 after treatment with HSP110 inhibitors (iHSP110, 10 µM) or control DMSO during 24 and 48 h (n = 3). E Immunoblot analysis of P-SYK, SYK, P-BTK BTK in BAJB after treatment with or without anti-IgM antibody (2.5 µg/mL) and with HSP110 inhibitor (iHSP110, 10 µM) or control DMSO during 48 h (n = 2). F Expression of MyD88, P-IκB and P-BTK by IHC in TMD8 tumor xenografted as in Fig. 1E (HE: Hematoxylin and Eosin), scale bar 40 µm.</figcaption></figure>

known to sustain the NFκB pathway through MyD88 binding and stabilization [31]. Accordingly, we observed by immunoprecipitation of MyD88 in HBL1 that both HSP110 inhibitors disrupted the interaction between HSP110 and MyD88, whereas compound #61 did not (Fig. 2C). The most frequent mutation of MyD88 (MyD88<sup>L265P</sup>) promotes the formation of a super complex, which contains BCR components and MyD88 [14]. Furthermore, BTK and SYK are both known to be activated by MyD88<sup>L265P</sup> in Waldestrom's macroglobulinemia and ABC-DLBCL [35, 36]. Therefore, we wondered if HSP110 inhibitors could impact components of the BCR pathway as part of this complex. We observed that HSP110 inhibitors reduce the phosphorylation of PLCy2, BTK and SYK in HBL1, OCI-Ly3 and TMD8 (Fig. 2D). To confirm this impact on the BCR pathway, we used BJAB, a Burkitt-like lymphoma cell line without BCR or TLR pathways mutations, that we stimulated with anti-IgM antibody. Reduced phosphorylation of BTK and SYK was also observed upon HSP110 inhibitor treatment (Fig. 2E). Immunohistochemistry staining of P-IκB and P-BTK in TMD8 xenografted tumors (from Fig. 1D) confirmed that those pathways were still strongly activated in vivo and that HSP110 inhibition markedly reduced their activation (Fig. 2F). Moreover, the disruption of HSP110/MyD88 interaction led to a decrease in MyD88 protein content. Taken together, our data suggest that HSP110 is necessary for optimal BCR and SYK activation and that it could be part of the MyD88 activation complex.

## HSP110 is a chaperone of SYK in the BCR pathway and promotes its activation

We then hypothesized that HSP110 could directly interact with either BTK or SYK as members of the initiation complex of MyD88 signaling. No interaction was detected between HSP110 and BTK (data not shown). In contrast, we observed an interaction between

HSP110 and SYK in TMD8, OCI-Ly3 (Fig. 3A, B), in HBL1 and U2932 (Fig. 3E). We confirmed, in cellulo, the proximity of the two proteins by PLA in TMD8 and OCI-Ly3 (Fig. 3C, D respectively). The number of HSP110-SYK binding spots was equal (TMD8) or even higher (OCI-Ly3) than in the PLA-positive control using antibodies against HSP110 and HSP70, two well-known binding partners. The specificity of the PLA experiments was confirmed by using a HSP110 siRNA, for which we lost most of the interaction (Fig. 3C, D). About 30% of ABC-DLBCL patients carry MyD88<sup>L265P</sup> mutations [6], which provides activation to SYK. So, we wondered if HSP110 interaction with SYK was dependent on MyD88<sup>L265P</sup> or whether it could also occur in cells with wild-type MyD88. Although smaller amounts of HSP110 and MyD88 were detected upon SYK immunoprecipitation in the U2932 cell line (MyD88wt, CD79wt) compared to the HBL1 cell line (MyD88L265P, CD79mut), SYK/HSP110 was still detected (Fig. 3E). We confirmed this in the HEK293 cell line transfected with wild-type MyD88, together with plasmid coding for SYK and HSP110 (Fig. 3F). We even found HSP110/SYK interaction in the non-stimulated BJAB cell line (MyD88wt, CD79wt), which was enhanced upon anti-IgM stimulation (Fig. 3G). Treatment of cells by iHSP110 strongly reduces the binding of HSP110 with SYK (Fig. 3H). TMD8-xenografted cells also show a significant interaction of HSP110 with SYK by PLA (Fig. 3I). It is worth noting that treatment with iHSP110 also disrupted in vivo interaction, as shown by the decrease in HSP110-SYK binding spots counted in each area of the tumor (Fig. 3I, J). Given the capacity of HSP110 to bind to STAT3 in colorectal cancer and to favor its phosphorylation by JAK2 [26], we hypothesized that HSP110 could also favor SYK phosphorylation. We thus performed an in vitro kinase assay with recombinant proteins. Our results showed that the autophosphorylation of SYK was increased in the presence of HSP110, and phosphorylation was abrogated in the presence of iHSP110 (Fig. 3K).
<figure><img src="image_10.png" /><figcaption>**Fig. 3** HSP110 is a chaperone for SYK and promotes its phosphorylation. Immunoprecipitation (IP) of HSP110 A and SYK B in TMD8 and OCI-Ly3 cells followed by immunoblot using anti-SYK A or anti-HSP110 B antibodies. A nonrelevant antibody was used as a control (IP control) (n = 3). Quantitation of in cellulo interactions of HSP110-HSP70 and HSP110-SYK in TMD8 C and OCI-Ly3 cells D by Duolink technology in the presence of siRNA HSP110 or siRNA control. HSP70 was used as a positive control for HSP110 interaction. Representative images of in cellulo interactions of HSP110-HSP70 and HSP110-SYK in TMD8 (C right) and in OCI-Ly3 (D right). Scale Bar 10 µm. E Immunoprecipitation (IP) of SYK in U2932 and HBL1 cells followed by immunoblot using anti-HSP110, anti-SYK, anti-MyD88 and anti-immunoglobulin heavy chains and anti-vinculin antibodies (n = 3). F Immunoprecipitation (IP) of SYK-FLAG in HEK293T cells transfected by HSP110-GFP, SYK-FLAG and MyD88-HA followed by immunoblot using anti-GFP, anti-FLAG, and anti-HA. (n = 3). G Immunoprecipitation (IP) of SYK in BJAB cells stimulated or not by anti-IgM (5 µg/ml) during 5 min and followed by immunoblot as in (G) (n = 3). H Immunoprecipitation (IP) of SYK in TMD8 and HBL1 cells followed by immunoblot using anti-HSP110, anti-SYK, and anti-immunoglobulin heavy chains and anti-vinculin antibodies. A nonrelevant antibody was used as a control (IP control). Cells were treated or not with iHSP110 20 µM during 24 h (n = 3). I In vivo quantitation of HSP110-SYK interaction in TMD8-derived tumor xenografted in NSG mice as in Fig. 1E. Mice were sacrificed at day 20 and HSP110-SYK interaction was determined on tumors by Duolink technology. Staining controls were done with either HSP110 antibody (Ab HSP110) or SYK antibody alone (Ab SYK), or no antibody (No Ab). J Representative Duolink images of Fig. 3I (scale bar 20 µm). K Immunoblot analysis of SYK, P-SYK and HSP110 after in vitro kinase assay performed in the presence of recombinant SYK as a substrate (200 ng) with or without recombinant HSP110 (10 ng), ATP (100 µM), and in the presence or not of iHSP110 (300 µM) (n = 3). ****P < 0.0001.</figcaption></figure>

To get clinical insight on the interaction between HSP110 and SYK, we performed PLA in biopsies from 15 patients with the ABC subtype and from 3 patients with the GC subtype. We confirmed a high level of HSP110/SYK interaction (Fig. 4A, B) and no significant differences between the ABC and GC subtypes (not shown).

To assess the importance of the reduction in BCR signaling by iHSP110 through the inhibition of SYK phosphorylation, we treated our cell lines with R406, a SYK-specific inhibitor [37], with or without iHSP110 (Supplementary Fig. 2A, B). We found that TMD8 viability is decreased by R406 and iHSP110. SYK phosphorylation was also reduced, and apoptosis markers were increased by inhibitors alone and in combination. This shows that TMD8 is sensitive to BCR-SYK signaling, which could partly explain the effect of iHSP110 on survival. OCI-Ly3, in contrast, had a limited dependency on SYK compared to iHSP110. To get more insight

into this, we expressed a hyperactive SYK mutant (TEL-SYK) [32] (Supplementary Fig. 2C) and measured OCI-Ly3 viability with or without iHSP110. Strong SYK activation abrogated the effect of R406 (Supplementary Fig. 2D) and reduced the effect of iHSP110 but without abrogation (Supplementary Fig. 2E). Taken together, our data confirmed the varying BCR dependency of DLBCL cells to SYK and suggest that the mechanism of action of iHSP110 is at least partially mediated by the SYK signaling pathway.

### Synergistic effect of iHSP110 and the phosphatidylinositol 3-kinase inhibitor copanlisib reduces xenografted tumor growth

The irreversible BTK inhibitor, ibrutinib, has shown some clinical benefit in ABC-DLBCL patients [19]. However, treatment-induced mutations such as BTK C481S confer resistance to the drug [38]. To
<figure><img src="image_11.png" /><figcaption>**Fig. 4 HSP110 and SYK interaction in DLBCL biopsies.** A Quantitation of in cellulo HSP110-SYK interaction in lymph node sections from 18 DLBCL patients. Controls of staining were done with either HSP110 antibody and isotype control antibody (HSP110-Ig), SYK antibody and isotype control antibody (Ig-SYK), or isotype control antibodies only (Ig-I). B Representative Duolink images of Fig. 4A (scale bar 20 µm). \*P < 0.05; \*\*P < 0.01.</figcaption></figure>

test whether iHSP110 may be an alternative in ibrutinib-induced resistant cells, we set up an experimental model of ibrutinib resistance by ectopic expression of either wild type BTK or BTK<sup>C4815</sup> in the ibrutinib-sensitive cell line TMD8. Transfection-induced expression of BTK<sup>C4815</sup> cells became totally resistant to ibrutinib in contrast to the cells expressing wild-type BTK (Supplementary Fig. 3A). Interestingly, treatment with iHSP110 suppressed cell growth equally in the three cell lines and reduced P-BTK expression (Supplementary Fig. 3B, C). This suggests that the effect of iHSP110 is independent of BTK status and, therefore, that targeting HPS110 may be a valuable alternative for Ibrutinib-resistant patients.

It is now well-established that ABC-DLBCL cells survival relies on two complementary pathways, the PI3K/AKT and NFκB pathways. The PI3K alpha isoform is highly expressed in ABC subtypes and most of the cell lines are addicted to this signaling [15, 39]. Treatment with the PI3Kα/δ inhibitor copanlisib synergized with the BCR-dependent NFκB inhibition by ibrutinib to remarkably suppress in vivo tumor growth. Because we found that iHSP110 inhibits BCR-dependent NFκB signaling through MyD88 and SYK, we hypothesized that a combined inhibition with copanlisib would result in a similar suppression of tumor growth. We therefore treated ABC-DLBCL cell lines with different doses of iHSP110 and/or copanlisib. Treatment with either drug alone inhibited cell line growth and viability, but the combination led to a synergistic suppressive effect using the Bliss independence model (Fig. 5A). Furthermore, the effective drug combination used in these experiments showed a strong decrease in NFκB, PI3K/AKT and BTK signaling, as well as in SYK phosphorylation (Fig. 5B). To confirm the efficacy of this combination in vivo, we treated a TMD8 xenograft in NSG mice every two other days with iHSP110 and/or copanlisib. We observed that while single treatments reduced tumor growth, the combination therapy almost completely abolished tumor growth (Fig. 5C, D). Accordingly, using IHC, we observed in these tumors that NFκB and BTK signaling were strongly reduced in vivo (Fig. 5E). Phospho-SYK was also reduced in vivo by combination of both inhibitors (Fig. 5F, G). Finally, we assessed the impact of the combinatorial treatment on tumor growth in a non-GC established patient-derived xenograft (PDX) mouse model. We observed a strong reduction in cell growth with copanlisib that was synergistically enhanced with iHSP110 (Fig. 5H). Overall, these findings suggest the interest of

combining our HSP110 inhibitor with PI3K/AKT pathway inhibitors in future clinical trials.

DISCUSSION

Over the last twenty years, conventional chemoimmunotherapy has considerably improved the survival rates of DLBCL patients. However, those with the aggressive ABC subtypes have the lowest clinical benefit because they are less responsive to therapy or because they relapse after an initial response. New therapeutic targets are urgently required for these patients. HSPs have been shown to be highly expressed in various cancers, including lymphomas. HSP90, the most abundant HSP, has been shown to be a critical hub to many pathways involved in tumor cell development, including the BCR signalosome and BCL6. However, while several HSP90 inhibitors have been tested in clinical trials in solid tumors [40], this approach has been disappointing due to the limited efficacy and unacceptable toxicity of those inhibitors. Among other HSPs with therapeutic potential, we and others have uncovered the chaperoning role of HSP110 for the master lymphoma-promoting proteins BCL6, C-MYC and MyD88. Developing drugs targeting HSP110 thus appears to be a very promising treatment strategy for lymphoma. In the present study, we used the first chemical molecule inhibitors of HPS110 developed by our team. Their specificity and anti-tumor properties have been validated in colorectal cancer models [30]. Here, we have demonstrated their capacity to alter the survival of several lymphoma cell lines in vitro and in vivo, and confirmed the inhibition of NFκB signaling. Because we monitored other critical survival signaling molecules, we observed a broader impact on the BCR pathway, which included BTK, PLCy2 and SYK. Crosstalk between the MyD88 and BCR pathways in ABC-DLBCL have been shown by the existence of a My-T-BCR supercomplex that encompasses the BCR components with MyD88<sup>L265P</sup>. Therefore, targeting MyD88 would lead to the alteration of the BCR pathway. We previously discovered that HSP110 interacted with MyD88<sup>WT</sup> as well as MyD88<sup>L265P</sup> to promote protein stability, which seemed to be reduced for the mutated MyD88 compared to the wildtype protein [31]. Our inhibitor abrogated the ability of HSP110 to interact with its chaperoned targets, as shown here by the disruption of HSP110/MyD88 interaction. MyD88 protein content should decrease over time upon treatment, which was indeed
<figure><img src="image_12.png" /><figcaption>**Fig. 5** HSP110 inhibitor and copanlisib synergize to decrease DLBCL cell survival. **A** Bliss synergy score of inhibitors combination in OCI-Ly3, TMD8 and HBL1. The numbers in the matrix indicate the antagonistic (below zero) or synergistic effect (above zero) of inhibitors (iHSP110 and copanlisib) on cell growth (n = 3). **B** Immunoblot analysis of P-SYK, SYK, P-AKT, AKT, P-BTK and BTK, in OCI-Ly3, TMD8 and HBL1 after treatment with HSP110 inhibitor (5 µM (OCI-Ly3 and TMD8) or 10 µM (HBL1)) and/or Copanlisib (3 nM (OCI-Ly3 and TMD8) or 25 nM (HBL1)) during 48 h (n = 3). **C** In vivo combination of iHSP110 and copanlisib treatment reduces tumor volume growth in mice. Tumor volume monitoring of TMD8 cells in NSG mice vehicle-treated or treated with the iHSP110 (10 mg/kg), copanlisib (5 mg/kg), or both. Animals were treated (i.p.) every 2 days. Mean volume (± SD) is represented (n = 6 in each group). **D** Tumor weight measured at day 21 after treatment as in (C). **E** Hematoxylin/eosin stain (HE) and expression of P-IkB and P-BTK by IHC in TMD8 tumor xenografted as in (C), scale bar 60 µm. **F** In vivo quantitation of P-SYK in TMD8-derived tumor xenografted in NSG mice as in Fig. 5C. Mice were sacrificed at day 21 and SYK/Phospho-tyrosine interaction was determined on tumors by Duolink technology. **G** Representative Duolink images of Fig. 5F, scale bar 20 µm. **H** Tumor volume monitoring of VFN-D1/KTC-bearing mice vehicle-treated or treated with the iHSP110 (10 mg/kg), copanlisib (5 mg/kg), or both. Animals were treated (i.p.) every 2 days. Mean volume (± SD) is represented (n = 6 in each group). \*P < 0.05; \*\*P < 0.01; \*\*\*P < 0.001; \*\*\*\*P < 0.0001.</figcaption></figure>

observed in long term in vivo treatment. Therefore, dramatic disorganization of the My-T-BCR supercomplex probably occurs after iHSP110 treatment, leading to the reduced activation of BCR components, subsequent failure of NFκB activation, and cell death. Since almost all patients with Waldenström macroglobulinemia have MyD88$^{L265P}$, they would likely also benefit from the use of our inhibitor to hamper the NFκB pathway.

In colorectal cancer, we have shown that HSP1110 interacts with STAT3 to promote its phosphorylation by JAK2, which is a very dynamic process. Here, we found that HSP110 interacts similarly with SYK to facilitate its phosphorylation. SYK is a key component of the BCR signaling pathway, which is activated by MyD88$^{L265P}$ in ABC-DLBCL and in WM cells [35]. Inhibition of SYK reduces the growth and survival of MyD88$^{L265P}$ lymphoma cells [2]. Furthermore, SYK activation promotes STAT3 and AKT signaling independently of the BCR pathway, adding to the importance of targeting this kinase to disrupt survival pathways besides NFκB [41]. We used the ability of SYK to autophosphorylate in our setting to demonstrate the promoting effect of HSP110. However, as the SRC family kinase LYN and HCK could serve as a trigger for SYK phosphorylation within the cells [42], it remains to be determined whether HSP110 cooperates with those two kinases.

When it comes to a new tumor target and drug that specifically neutralizes its function, we must consider a combination with approved treatments to either improve efficacy or overcome induced resistance. For instance, ibrutinib shows great efficacy in patients harboring both MyD88 and CD79a mutations [19], but

resistance may arise due to mutations in cysteine residues in the active site of BTK. By introducing BTK$^{C481S}$ in a DLBCL-sensitive cell line, we showed that iHSP110 activity in ABC-DLBCL tumors is not impacted by this mutation. It could therefore potentially be used as an alternative drug in Ibrutinib-induced resistant cells. However, since many of these cells do not have BTK mutations but rather activation of other survival pathways, or have an alternative activator of the BTK pathway such as the GTPase RAC2 [43], it may be worth determining whether iHSP110 also affects RAC2 activation.

Besides Ibrutinib and the BTK pathway, the PI3K/AKT pathway has been recently identified as an important alternative survival pathway [15, 39, 44]. Copanlisib, a pan class I PI3K inhibitor, has demonstrated, as a sole agent in relapsed/refractory DLBCL, as much efficacy as ibrutinib [45], with ABC-DLBCL being the best responding DLBCL subtype. In addition, PDX models treated with a combination of ibrutinib and copanlisib show impressive tumor reductions [15]. Here we showed that combining iHSP110 with copanlisib also has a strong anti-tumor effect in vitro and in vivo. This could be explained by a broad effect on most of the survival pathways described so far in ABC-DLBCL. Indeed, (a) upon iHSP110, the My-T-BCR supercomplex is dislocated following the disappearance of MyD88, SYK is less phosphorylated and the subsequent BCR pathway is less activated, and (b) upon copanlisib treatment, PI3Kα and δ are neutralized, blunting the downstream pathways. Several other PI3K inhibitors are in preclinical and clinical development. Our data suggest the relevance of testing iHSP110 in association with the PI3K inhibitor that proves to be the most efficient. The patients that would benefit the most from
this treatment have not yet been identified since molecular data to predict response are still lacking. We have shown that all patients express HSP110 and that more than 80% of cells are positive, suggesting that HSP110 expression is not a limiting factor at diagnosis [31]. Little is known about changes in HSP110 expression in tumors over the course of the disease and chemotherapy, but this question warrants further investigation.

Our inhibitors are the first-in-class in targeting HSP110 and we must pay attention to potential unwanted side effects of HSP110 inhibition. iHSP110 has shown no toxicity in mice models and HSP110⁻¹ mice have normal development [46]. But, giving the importance of HSP110 in the prevention of neurodegenerative diseases in mice, further investigation must be carried out [47]. We show here their potential to significantly reduce DLBCL tumor growth through MyD88 and SYK inhibition, two critical components of the MY-T-BCR signaling complex. In addition, tumor growth can be further repressed in association with PI3K inhibition. We intend to improve their pharmacological properties by chemistry optimization to foster preclinical development, as we believe that HSP110 inhibition is a promising therapeutic tool for DLBCL treatment, either alone or in combination with other targeted therapies.

## DATA AVAILABILITY

Full materials, methods and data may be shared after request to the corresponding author by e-mail gaetan.jego@u-bourogne.fr.

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## ACKNOWLEDGEMENTS

This work was supported by grants from the Ligue Nationale Contre le Cancer EL2023 LNCC/CaG (GJ and CG), from the Fondation ARC pour la recherche sur le Cancer (GJ), the French National Research Agency (ANR) under the program "Investissements d'Avenir" with reference ANR-11-LABX-0021 (LabEX LipSTIC), ANR-11-LABX-0051 (Labex GR-Ex) and ANR-15-IDE-0003 (I-SITE-BFC), the Conseil Regional de Bourgogne, We also thank the FEDER for their support. This work was also possible with help from Flow Cytometry Core Facility/Inserm UMR1231, at the University of Burgundy.

## AUTHOR CONTRIBUTIONS

VCG, MD, CB, FH, KN, PK and GJ designed the study and experiments; VCG, MD, CB, FH, KN, and MA performed the experiments; CCC and CG provided samples and expertise; VCG, MD, CB, FH, CCC, MA, PK, CG, GJ analyzed the experiments; GJ directed the work.

## ETHICS APPROVAL AND CONSENT TO PARTICIPATE

Research using human material was done in accordance with the Declaration of Helsinki and was approved by Centre Leon Berard ethics committee, Lyon, France (reference AC-2019-3426). All Human participants gave informed consent.

## COMPETING INTERESTS

The authors declare no competing interests.

## ADDITIONAL INFORMATION

**Supplementary information** The online version contains supplementary material available at https://doi.org/10.1038/s41375-024-02302-x.

**Correspondence** and requests for materials should be addressed to Gaëtan Jego.

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