www.nature.com/onc Oncogene ARTICLE OPEN Targeting the MYC interaction network in B-cell lymphoma via histone deacetylase 6 inhibition René Winkler1,9,10, Ann-Sophie Mägdefrau 1,10, Eva-Maria Piskor 1, Markus Kleemann 1, Mandy Beyer2, Kevin Linke 1, Lisa Hansen1, Anna-Maria Schaffer 1, Marina E. Hoffmann 3, Simon Poepsel 4,5, Florian Heyd6, Petra Beli 3, Tarik Möröy7, ✉ Siavosh Mahboobi8, Oliver H. Krämer 2 and Christian Kosan 1 © The Author(s) 2022 Overexpression of MYC is a genuine cancer driver in lymphomas and related to poor prognosis. However, therapeutic targeting of the transcription factor MYC remains challenging. Here, we show that inhibition of the histone deacetylase 6 (HDAC6) using the HDAC6 inhibitor Marbostat-100 (M-100) reduces oncogenic MYC levels and prevents lymphomagenesis in a mouse model of MYC- induced aggressive B-cell lymphoma. M-100 specifically alters protein-protein interactions by switching the acetylation state of HDAC6 substrates, such as tubulin. Tubulin facilitates nuclear import of MYC, and MYC-dependent B-cell lymphoma cells rely on continuous import of MYC due to its high turn-over. Acetylation of tubulin impairs this mechanism and enables proteasomal degradation of MYC. M-100 targets almost exclusively B-cell lymphoma cells with high levels of MYC whereas non-tumor cells are 1234567890();,: not affected. M-100 induces massive apoptosis in human and murine MYC-overexpressing B-cell lymphoma cells. We identified the heat-shock protein DNAJA3 as an interactor of tubulin in an acetylation-dependent manner and overexpression of DNAJA3 resulted in a pronounced degradation of MYC. We propose a mechanism by which DNAJA3 associates with hyperacetylated tubulin in the cytoplasm to control MYC turnover. Taken together, our data demonstrate a beneficial role of HDAC6 inhibition in MYC-dependent B-cell lymphoma. Oncogene (2022) 41:4560–4572; https://doi.org/10.1038/s41388-022-02450-3 INTRODUCTION and the absence of druggable structures make MYC a difficult B-cells are prone to lymphomagenesis due to their high target for direct inhibition [11]. Attempts to directly target MYC via proliferative capacity and dependence on physiological DNA small molecules did not achieve adequate results as these drugs damage during V(D)J recombination and affinity maturation in underwent rapid degradation and showed unfavorably high IC50 germinal centers [1]. Non-Hodgkin’s lymphomas, such as Burkitt’s values [11]. In fact, physiological levels of MYC fulfill crucial lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL) are functions in many cell types, and pharmacological targeting of aggressive heterogeneous lymphomas originating from germinal MYC should only counteract supraphysiological MYC levels center B-cells [2]. BL is typically characterized by translocations of present in malignant cells. MYC to the vicinity of potent immunoglobulin enhancers, such as MYC can recruit epigenetic modifiers, such as the histone t(8;14) [1]. DLBCL shows various molecular alterations, among acetyltransferases p300/CBP or the histone deacetylases (HDACs) them translocations of BCL6 or BCL2 [1]. However, MYC transloca- HDAC1 and HDAC3 to activate or repress distinct genes in cancer tions also occur in around 15% of DLBCL [2], and elevated cells [8]. Pan-HDAC inhibitors (pan-HDACi) that target several expression of MYC correlates with poor clinical prognosis in B-cell HDACs have been shown to give promising results in hematologic lymphoma [3, 4]. Overexpression of the transcription factor MYC malignancies [12–14]. The regulation of non-histone proteins, in leads to fatal misregulation of cellular metabolism, cell growth, particular proto-oncogenes, by HDACi enables the control of many and signaling pathways [5, 6]. Moreover, MYC controls prolifera- essential cellular processes, such as cell survival, proliferation, tion, angiogenesis, and mRNA processing in tumor development protein stability, and protein interactions [15]. For example, pan- [7, 8]. HDACi treatment has been shown to inhibit BCL-6 function by MYC is known to associate with microtubules for the nuclear stabilizing BCL-6 acetylation, which leads to the de-repression of import of MYC [9]. Importantly, MYC has a half-life of roughly half its target genes [16]. Interestingly, MYC can be found acetylated at an hour when transiently expressed [10]. This high turn-over rate K423 upon pan-HDACi treatment, which decreases MYC 1 Department of Biochemistry, Center for Molecular Biomedicine (CMB), Friedrich Schiller University Jena, Jena 07745, Germany. 2Institute of Toxicology, University Medical Center Mainz, Mainz 55131, Germany. 3Institute of Molecular Biology (IMB), Mainz 55128, Germany. 4Center for Molecular Medicine Cologne (CMMC), Faculty of Medicine and University Hospital, University of Cologne, Cologne 50931, Germany. 5Cologne Excellence Cluster for Cellular Stress Responses in Ageing-Associated Diseases (CECAD), University of Cologne, Cologne 50931, Germany. 6Laboratory of RNA Biochemistry, Institute of Chemistry and Biochemistry, Freie Universität Berlin, Berlin 14195, Germany. 7Institut de Recherches Cliniques de Montréal (IRCM), Montréal, QC H2W 1R7, Canada. 8Department of Pharmaceutical/Medicinal Chemistry I, Institute of Pharmacy, University of Regensburg, Regensburg 93040, Germany. 9Present address: Josep Carreras Leukaemia Research Institute (IJC), Campus ICO-Germans Trias i Pujol, Badalona 08916, Spain. 10These authors contributed equally: René Winkler, Ann-Sophie Mägdefrau. ✉email: christian.kosan@uni-jena.de Received: 12 February 2022 Accepted: 22 August 2022 Published online: 6 September 2022 R. Winkler et al. 4561 transcription via autoregulation and results in apoptosis [13]. M-100-treated mice manifested a lymphoma (B220+ IgM-) and However, the success of pan-HDACi in pre-clinical studies only had to be sacrificed. Lymphomas from non-treated and vehicle- partially improved treatment for patients with hematologic treated Eµ-Myc mice showed a 2:1 ratio of IgM- to IgM+ B-cell malignancies [17]. tumors indicating that lymphoma cells in this model system have The use of HDACi that target singular members of the HDAC an immature or mature B-cell origin (Fig. 1D), as observed before family will help to understand separate HDAC functions in [26]. Long-term treatment with M-100 did not affect the body hematologic malignancies. For example, treatment of DLBCL cells weight of mice (Supplemental Fig. 2D). The mean spleen weight of with the HDAC6 inhibitor ACY-1215 (Rocilinostat) was shown to Eµ-Myc mice, however, was significantly decreased after contin- activate the unfolded protein response by increasing quantity and uous M-100 treatment, indicating reduced disease progression acetylation of heat-shock proteins (HSPs), eventually resulting in (Fig. 1E, Supplemental Fig. 2E, F). We analyzed immune cell cell death [18]. A first clinical trial involving treatment of populations of surviving mice by flow cytometry. M-100-treated lymphoma patients with ACY-1215 was completed and stated a Eµ-Myc mice showed a slightly reduced splenic CD19+ B-cell pool favorable safety profile [19]. (Fig. 1F, Supplemental Fig. 3A). B-cell development in the bone Interestingly, HDAC6 represents a microtubule-associated dea- marrow was despite this not affected by M-100 treatment cetylase and was shown to deacetylate microtubules at K40 (Supplemental Fig. 3B) and increased apoptosis of B-cells was [20, 21]. Recently, a novel HDAC6 inhibitor, Marbostat-100 (M-100), not detected in vivo (Supplemental Fig. 3C). In fact, B220low cells was developed based on the structure of Tubastatin A [22, 23]. were still present in spleens (Supplemental Fig. 3C), which have M-100 inhibits the major catalytic domain of HDAC6 with at least been previously described as pre-tumor B-cells in this model 250-fold higher affinity compared to other HDACs and the binding [27, 28]. Besides, survivors from the M-100 cohort showed a mode was well described [22, 23]. Moreover, M-100 was well- significant decline in CD11b+ myeloid cells and an absolute as tolerated in a preclinical model of rheumatoid arthritis and well as a relative increase in CD3+ T-cells compared to the vehicle showed favorable pharmacodynamics in mice [22]. group (Fig. 1F, Supplemental Fig. 3A). Here, further investigation Our findings identified the HDAC6 inhibitor M-100 as a new tool showed that survival of primary CD4+ and CD8+ T-cells was not to target MYC-dependent lymphomas. M-100 efficiently pre- affected by M-100 treatment, whereas proliferation of CD4+ T-cells vented lymphomagenesis, and induced apoptosis in human and after M-100 treatment was even increased when cultured together murine lymphoma cells by reducing high MYC levels without with B-cells (Supplemental Fig. 3D–F). These results suggest that harming untransformed B-cells. On a molecular basis, we M-100 affects myeloid cell abundance and T-cell proliferation identified a novel cytoplasmic interaction network formed by which might have contributed to prevent B-cell lymphomagenesis tubulin, HDAC6, and HSPs that regulates the protein stability of in Eµ-Myc mice. MYC. Our results suggest that inhibition of HDAC6 can be used to Next, we assessed the curative potential of M-100 on already target excessive MYC in cancer. manifested lymphoma. We treated lymphoma-bearing Eµ-Myc mice with M-100 and analyzed their survival. After the detection of lymphoma, untreated Eµ-Myc mice died within 6 days. Acute RESULTS M-100 treatment of sick Eµ-Myc mice significantly improved HDAC6 inhibition increases the survival of lymphoma-prone survival almost 3-fold, which was also indicated by maintaining mice lower disease severity scores (Fig. 1G, Supplemental Fig. 4). Taken The Eµ-Myc mouse line is a commonly used model for studying together, HDAC6 inhibition strongly reduced B-cell lymphoma- the spontaneous formation of B-cell lymphomas due to B-cell- genesis and extended life span in mice overexpressing MYC in the specific overexpression of MYC, resembling partially disease B-cell compartment. phenotypes of BL or DLBCL [24]. We isolated lymphoma B-cells from Eµ-Myc mice and treated these cells ex vivo with the HDAC6 Inhibition of HDAC6 specifically induces apoptosis in B-cell inhibitor M-100. Interestingly, mouse lymphoma cells showed a lymphoma dose-dependent reduction of Myc protein levels after M-100 To test the effects of HDAC6 inhibition on lymphoma B-cells treatment (Fig. 1A). We also detected a strong acetylation of the ex vivo, purified lymphoma cells from Eµ-Myc mice were treated HDAC6 substrate tubulin concluding efficient HDAC6 inhibition for 72 h with increasing amounts of M-100. Purified B-cells from (Fig. 1A), which is a striking result as M-100 was originally wild-type mice were activated with 10 µg/ml LPS and served as developed against human HDAC6 [22, 23]. However, levels of control. B-cells from Eµ-Myc mice showed a strong induction of unmodified tubulin remained unaltered (Supplemental Fig. 1). apoptosis already after treatment with 1 µM M-100 (Fig. 2A). Of Besides, Parp-1 cleavage was detected in M-100-treated mouse note, activated B-cells from wild-type mice did not show an lymphoma cells, indicating apoptosis induction (Fig. 1A). To test increased apoptosis rate (Fig. 2A). We also assessed the effect of the effect of M-100 in vivo, we applied M-100 at 30 mg/kg as M-100 on the cell cycle of lymphoma and activated wild-type B- described before [22] via intraperitoneal (i.p.) injection to C57BL/6 cells. Murine lymphoma cell populations showed a significant mice. M-100 treatment did not affect immune cell populations in increase of cells in the subG1 fraction, indicating apoptosis, and mice (Supplemental Fig. 2A–C), as previously shown [22]. a reduction in G1/S-phases 72 h after M-100 treatment Acetylation of tubulin was increased in splenic cells 6 h after (Fig. 2B, C). However, non-malignant B-cells did not any show injection and declined until 72 h post treatment (Fig. 1B). These cell cycle alterations (Fig. 2B, C). Gene expression of Bbc3 and results prompted us to test if continuous treatment with M-100 Pmaip1, encoding pro-apoptotic Puma and Noxa was increased could prevent lymphoma development in Eµ-Myc mice. in lymphoma but not wild-type B-cells after M-100 treatment To study the long-term effect of HDAC6 inhibition by M-100 on (Fig. 2D). Conversely, transcription of Bcl2 was strongly B-cell lymphomagenesis, 70-day-old Eµ-Myc mice with a high decreased in lymphoma cells after HDAC6 inhibition (Fig. 2D), incidence for lymphoma development [25], received i.p. injections which underlines the apoptotic phenotype (Fig. 2A–C). M-100 with 30 mg/kg M-100 or vehicle every 72 h for six weeks. Mice also affected Myc protein levels in activated wild-type B-cells were monitored for an additional period of six weeks for that was not caused by transcriptional changes (Fig. 2D, E). spontaneous tumor formation after M-100 withdrawal. M-100 However, wild-type B-cells showed a strong upregulation of the treatment of Eµ-Myc mice significantly increased the overall anti-apoptotic factor Bcl-2 after M-100 treatment that was not survival compared to vehicle-treated and untreated mice, even present in Eµ-Myc cells, and which likely protected wild-type B- after M-100 withdrawal (Fig. 1C). While 60% of vehicle-treated Eµ- cells from apoptosis induction (Fig. 2E). To further examine the Myc mice developed lymphomas in this period, only one out of 17 role of Bcl-2, we treated proliferating B-cells from wild-type mice Oncogene (2022) 41:4560 – 4572 R. Winkler et al. 4562 A M-100 B Intraperitoneal injection 0 1 2 4 [μM] Vehicle 6 72 [h] Myc 70 kDa M-100 (30 mg/kg) 6 6 72 72 [h] 1.00 0.56 0.60 0.65 Ac-tub 55 kDa Ac-tub 55 kDa 130 kDa 100 kDa Vinculin Cl. Parp-1 Mouse wild-type splenocytes Actin 40 kDa Eμ-Myc lymphoma cells ex vivo C D Lymphoma phenotype E P=0.047 Tx Monitoring 800 P=0.025 Number of Eμ-Myc mice 40 IgM- 100 Spleen weight [mg] IgM+ 600 P=0.951 30 Mixed Survival [%] 400 50 Eμ-Myc mice 20 No Tx, n=46 P<0.001 200 M-100 (30 mg/kg), n=17 10 P=0.001 Vehicle, n=15 vehicle, 0 0 0 50 100 150 e 00 pe Tx e 00 cl Lifespan [days] cl -1 - ty -1 hi hi o M N Ve M Ve ild W F Survivor analysis (spleen) G CD19+ cells CD3+ cells CD11b+ cells P=0.101 P=0.013 20 P<0.001 Eμ-Myc mice with lymphoma burden 50 70 60 100 No Tx, n=15 15 P=0.002 M-100 (30 mg/kg), n=5 40 Survival [%] 50 [%] 10 [%] [%] 40 50 30 5 30 20 20 0 0 0 5 10 15 20 25 00 00 le 00 le e cl ic ic -1 -1 -1 Lifespan [days] hi h h M M Ve M Ve Ve Fig. 1 Continuous HDAC6 inhibition increases survival of lymphoma-prone MYC-overexpressing mice. A Western Blot analysis of Eµ-Myc lymphoma cells isolated from malignant lymph nodes and treated ex vivo with increasing concentrations of M-100 for 24 h. Levels of Myc were quantified to untreated conditions. Actin was used as a loading control. Cl. - cleaved. B Western Blot analysis of splenocytes from wild- type mice treated once via i.p. injection with 30 mg/kg M-100 or vehicle for the indicated time. Each lane represents one individual mouse. Vinculin was used as a loading control. Ac-tub - acetylated tubulin. C Survival curves of Eµ-Myc mice treated every 72 h with 30 mg/kg M-100 (n = 17) or vehicle (n = 15). Treatment (Tx) started at age 70 d for six weeks. Mice were monitored for additional six weeks for signs of lymphoma development. Median survival vehicle cohort: 136 d, M-100 cohort: not possible to calculate, untreated Eµ-Myc mice: 140 d. Log- Rank-test. D Lymphoma phenotypes of diseased Eµ-Myc mice measured by flow cytometry. E Spleen weights of all mice from the different cohorts. Spleen weights from wild-type mice serve as control. Wilcoxon rank-sum test. F Flow cytometry analysis of splenic immune cell populations of survivors. Unpaired Student’s t-test, two-tailed. G Survival curves of Eµ-Myc mice suffering from lymphoma treated with M-100 (median 16 d) or left untreated (median 6 d). Mice received M-100 (30 mg/kg) every 72 h starting when palpable lymph node swelling was present. Treatment continued until endpoint criteria were reached. Log-Rank-test. Data in A are representative of n = 3 independent experiments. E, F Each dot represents one mouse. Bars depict mean. with the highly specific BCL-2 inhibitor Venetoclax (Fig. 2F). HDAC6 inhibition results in apoptosis of human B-cell Inhibition of Bcl-2 in combination with M-100 led to a strong lymphoma cells apoptosis induction of activated B-cells, which indicates that We also assessed the response to M-100 in human B-cell upregulation of Bcl-2 mediated survival signals in healthy B-cells lymphoma cell lines of the BL and DLBCL type characterized by (Fig. 2F). We also checked the effects of M-100 on CH12F3 cells, a MYC translocation or amplification [30–35], although the indivi- murine B-cell line harboring no MYC translocation [29]. In line dual MYC mutation profile differed greatly in these cells [36, 37], with the findings from wild-type B-cells, no apoptosis or altered (Table 1). We determined the efficacy of M-100 by MTT assay, and cell cycle was measured after M-100 treatment in CH12F3 cells four out of five tested cell lines showed striking dose-responses to (Fig. 2G). Taken together, M-100 exclusively induced apoptosis M-100 (Fig. 3A). The obtained IC50 values ranged between 2.07 µM in murine lymphoma cells with elevated Myc levels as these cells and 5.25 µM. Raji cells, however, were less sensitive to M-100 failed to upregulate Bcl-2. (IC50 = 17.17 µM), although a moderate apoptosis induction was Oncogene (2022) 41:4560 – 4572 R. Winkler et al. 4563 A B Eμ-Myc lymphoma cells M-100 0 μM M-100 CTR 1 2 4 [μM] 5.4 19.8 31.1 60.6 4 μM M-100 Eμ-Myc lymphoma cells subG1 0.7 0.9 1.4 1.0 Activated B-cells 4.7 2.4 3.0 5.4 0 μM M-100 Activated B-cells (WT) PI Frequency 3.6 2.4 2.4 3.0 4 μM M-100 subG1 Annexin V C D PI Eμ-Myc Activated lymphoma cells B-cells Puma (Bbc3) Noxa (Pmaip1) Bcl2 Myc P<0.001 P=0.016 P<0.001 P=0.160 100 ns G2M 4 10 1.5 4 ns ns S 8 3 3 G1 Fold induction 1.0 * subG1 6 [%] 50 2 2 ns 4 0.5 * 1 2 1 ns 0 *** 0 0 0.0 0 4 μM M-100 + + 4 μM M-100 + + + + + + + + Eμ-Myc WT Eμ-Myc WT Eμ-Myc WT Eμ-Myc WT E M-100 F G 0 1 2 4 [μM] Activated B-cells CH12F3 cells 25 kDa Bcl-2 P<0.001 Actin 15 80 100 40 kDa Annexin V+ PI+ cells [%] ns ns Eμ-Myc lymphoma cells P=0.026 60 ns Viability [%] #1 #2 #3 10 P=0.026 ns 4 μM M-100 + + + [%] 40 50 70 kDa Myc 5 ns ns 20 Ac-tub 55 kDa 25 kDa ns Bcl-2 0 0 ns 0 BCL-2i + + Activation + + + + Gapdh 35 kDa M-100 + + M-100 + + Activated B-cells Fig. 2 M-100 specifically induces apoptosis in murine lymphoma B-cells but not activated wild-type B-cells. A Apoptosis detection using Annexin V and PI staining. Eµ-Myc lymphoma and activated (10 µg LPS/ml) wild-type mouse B-cells were compared. B Representative cell cycle analysis of Eµ-Myc lymphoma and activated B-cells treated for 72 h. C Cell cycle analysis from B was quantified. Two-Way ANOVA (Sidak’s posthoc). D Gene expression changes of Eµ-Myc lymphoma or activated wild-type (WT) B-cells cells treated for 24 h with 4 µM M-100 using quantitative real-time PCR analysis. One-Way ANOVA (Tukey’s posthoc). E Western Blot analysis of activated wild-type B-cells or Eµ-Myc lymphoma cells treated with M-100 for 24 h. Gapdh and actin were used as loading controls. Ac-tub - acetylated tubulin. F Apoptosis was analyzed in activated wild-type B-cells treated with 4 µM M-100, 0.5 µM BCL-2 inhibitor (BCL-2i) Venetoclax or both for 24 h. One-Way ANOVA (Tukey’s posthoc). G CH12F3 cells were analyzed regarding viability (Annexin V/PI staining) and cell cycle after treatment with 2 µM M-100 for 48 h. Activation was performed using 1 µg/ml CD40L, 5 ng/ml IL-4, and 1 ng/ml TGF-β and added 2 h after M-100 treatment. CH12F3 cells harbor no MYC translocation. Two-Way ANOVA (Sidak’s posthoc). Data in A–G are representative of at least n = 3 independent experiments. Data represent mean + SEM, if applicable. *P < 0.05, ***P < 0.001, ns - not significant. Oncogene (2022) 41:4560 – 4572 R. Winkler et al. 4564 our previous findings from murine lymphoma cells, we also Table 1. MYC mutations in human B-cell lymphoma cell lines. detected PARP-1 cleavage 24 h after treatment with M-100 Cell line MYC mutations Type (Fig. 4A). On the contrary, BCL-2 levels decreased after HDAC6 Ramos° Translocated BL inhibition although transcription of the BCL2 gene was elevated, assuming a possible feedback loop (Fig. 4A, Supplemental Raji Translocated; p.YQ32del, p.S6T, p.E39D, BL Fig. 7B, C). Expression of BIM, encoding a pro-apoptotic mediator, p.A44V, p.T58I was significantly upregulated which was also present at the BL-30 Translocated; p.Q50H, p.T73I, p.G105D, BL protein level (Supplementary Fig. 7B, C). As in murine lymphoma p.S218N cells, the decrease of MYC was not due to transcriptional changes SUDHL-6 Translocated DLBCL (Supplementary Fig. 7C). To determine if MYC degradation is OCI-Ly3 Amplified DLBCL regulated by ubiquitin-mediated proteolysis upon HDAC6 inhibi- Table summarizing MYC mutations in the used cell lines from the CCLE and tion, we performed combinatorial treatments with M-100 and the COSMIC databases. °Sequencing data were only available for subclone proteasome inhibitor MG132 (Fig. 4B). MYC degradation was Ramos2G64C10. BL Burkitt’s lymphoma, DLBCL diffuse large B-cell efficiently blocked when proteasomes were not functioning. The lymphoma. use of MG132 resulted in pronounced general accumulation of ubiquitinated proteins, in contrast to M-100 (Fig. 4C). In addition, immunoprecipitation (IP) of MYC after HDAC6 inhibition revealed achieved at 4 µM (Fig. 3B). M-100 treatment resulted in the ubiquitinated MYC protein bands at molecular weights of 85 kDa induction of apoptosis in all tested human BL and DLBCL cell lines and 100 kDa (Fig. 4C). In fact, all tested BL and DLBCL cell lines as demonstrated by the accumulation of Annexin V+ cells (Fig. 3B). showed a substantial decrease of MYC protein after 6 h treatment Consistent with our findings from murine lymphoma cells, we with M-100 (Fig. 4D), pointing towards a general mechanism of detected a significant increase of subG1 fractions 48 h post M-100 MYC degradation after HDAC6 inhibition. We also observed a treatment (Fig. 3C), and a reduced entry into S-phase already 24 h slight increase in MYC turnover when HDAC6 was knocked out post-treatment in human lymphoma cells (Supplemental Fig. 5A- (Supplementary Fig. 8). These results indicate that HDAC6 C). Only BL-30 cells showed an unaltered cell cycle profile, which inhibition by M-100 specifically provokes ubiquitination of MYC might be explained by the high mutation rate within the MYC and subsequent proteasomal degradation. gene (Fig. 3C, Table 1). To confirm apoptosis as the responsible cell death mechanism in Ramos cells, combinatorial treatment of MYC degradation is associated with changes in the M-100 and the caspase-specific inhibitor Z-VAD-FMK was interactome of hyperacetylated tubulin performed (Supplemental Fig. 5D). The use of both Z-VAD-FMK To test whether the proteasomal degradation of MYC is mediated by and M-100 prevented PARP-1 and Caspase 3 cleavage and a direct interaction, we performed IPs and were able to pull-down ultimately reduced the number of early apoptotic cells (Supple- endogenous HDAC6/MYC complexes in Ramos cells (Fig. 5A). These mental Fig. 5E). These results clearly show that cell death complexes also appeared when the catalytic activity of HDAC6 was induction by M-100 is facilitated via the apoptotic pathway. inhibited. However, HDAC6 is localized exclusively in the cytoplasm, To exclude off-target effects of M-100, we compared different whereas MYC is found predominantly in the nucleus (Fig. 5B). Thus, concentrations of M-100 to the pan-HDACi MS-275 that inhibits we analyzed the impact of MYC localization for its degradation. For HDAC1, HDAC2, and HDAC3. H3 acetylation was induced at this purpose, we transfected NIH-3T3 cells to express GFP-MYC and concentrations greater than 6 µM M-100 (Fig. 3D). Importantly, this treated these cells with Importazole or Leptomycin B to block nuclear limit is above most obtained IC50 values for M-100 in B-cell import or export, respectively (Fig. 5C). Afterward, cells were treated lymphoma cells, suggesting a specific effect of M-100 (Fig. 3A). with cycloheximide (CHX) to induce cell-intrinsic degradation and Furthermore, M-100 had superior targeting properties compared levels of MYC-GFP were tracked via flow cytometry. Interestingly, the to the HDAC6 inhibitor ACY-1215 as HDAC6 was efficiently degradation of MYC-GFP was significantly accelerated in the inhibited by M-100 but HDACs that deacetylate histone H3 were cytoplasm but not in the nucleus (Fig. 5C). not (Supplemental Fig. 6A). To further validate our findings, we We wanted to analyze the localization of MYC in response to generated Ramos HDAC6 knock-out (KO) cells using targeted HDAC6 inhibition by M-100. For this purpose, we treated Ramos CRISPR/Cas9 technology. HDAC6 KO cells were characterized by cells that harbor very little cytoplasmic compartment with M-100 permanent hyperacetylation of tubulin (Fig. 3E). Importantly, KO of and used proximity ligation assay (PLA). We detected a close HDAC6 mimics to some extent the treatment with M-100 as localization between MYC and acetylated tubulin under physio- Ramos HDAC6 KO cells showed reduced proliferation and logical conditions where a small amount of acetylated tubulin is increased apoptosis compared to WT cells (Fig. 3F, Supplemental present (Fig. 5D). However, PLA signals disappeared after M-100 Fig. 6B). As expected, Ramos HDAC6 KO cells were rather treatment when tubulin was hyperacetylated (Fig. 5D). It had been insensitive to M-100 treatment (IC50 = 9.47 µM) as measured with previously described that MYC associates with tubulin [9] and our MTT assay and apoptosis detection by Annexin V/PI staining results indicate that this MYC-tubulin interaction is acetylation- (Fig. 3G, Supplemental Fig. 6B). However, KO of HDAC6 barely dependent. altered the response of Ramos cells towards ACY-1215, revealing a To further investigate the effect of hyperacetylated tubulin, a very narrow window of specific targeting (Fig. 3G, Supplemental detailed interactome analysis of hyperacetylated tubulin was Fig. 6B). This underlines that M-100 has improved specificity for performed in MYC-dependent blood cancer cells via mass HDAC6 compared to ACY-1215 whose off-targets were recently spectrometry. Bound proteins were identified and considered by described in detail [38]. either increased abundance (>2-fold) or new binding to tubulin after M-100 treatment compared to control (Supplementary HDAC6 inhibition causes MYC degradation Table 1). From 2309 identified proteins, treatment with M-100 Human MYC-overexpressing lymphoma cells responded to M-100 led to enhanced binding of 357 proteins and new binding of 323 treatment with a strong induction of apoptosis. To shed further proteins to hyperacetylated tubulin (Fig. 5E). A functional light on the underlying cellular mechanism, we treated Ramos annotation of the altered tubulin interactome after M-100 cells with different concentrations of M-100 for 6 h and 24 h. MYC treatment using the DAVID tool confirmed significant changes in protein levels were already reduced 6 h after treatment with 2 µM proteins related to acetylation, nucleotide-binding, protein trans- and 4 µM M-100 (Fig. 4A, Supplemental Fig. 7A). Consistent with port, and ubiquitin conjugation (Fig. 5E). Overrepresented protein Oncogene (2022) 41:4560 – 4572 R. Winkler et al. 4565 A Ramos (BL) Raji (BL) BL-30 (BL) SUDHL-6 (DLBCL) OCI-Ly3 (DLBCL) 100 100 100 100 100 Viability [%] 50 50 50 50 50 IC50=3.37 IC50=17.17 IC50=4.99 IC50=5.25 IC50=2.07 0 0 0 0 0 0.01 0.1 1 10 0.01 0.1 1 10 0.01 0.1 1 10 0.01 0.1 1 10 0.01 0.1 1 10 M-100 [μM] M-100 [μM] M-100 [μM] M-100 [μM] M-100 [μM] B Ramos Raji BL-30 SUDHL-6 OCI-Ly3 P=0.009 P=0.001 P=0.012 P=0.005 P=0.005 60 15 60 20 80 Annexin V+ cells [%] 15 60 40 10 40 10 40 20 5 20 5 20 0 0 0 0 0 4 μM M-100 + + + + + C Ramos Raji BL-30 SUDHL-6 OCI-Ly3 100 100 100 100 100 G2M ns ns *** * ns S ns * G1 ** * subG1 ns ns [%] 50 50 50 50 50 ns *** ** ns ns ns ** *** 0 ** 0 0 0 0 4 μM M-100 + + + + + D M-100 MS-275 E F CTR 2 4 6 8 10 5 [μM] Ac-tub 55 kDa P=0.002 HDAC6 130 kDa 15 kDa 100 HD6 WT Ac-H3 Ac-tub 55 kDa 130 kDa HD6 KO Vinculin 80 Proliferation [%] Actin 40 kDa Ramos Ramos 60 G 40 100 100 20 Viability [%] Viability [%] 0 50 50 24 48 72 [h] KO, IC50=9.47 KO, IC50=2.60 WT, IC50=3.37 WT, IC50=1.87 0 0 0.1 1 10 0.1 1 10 M-100 [μM] ACY-1215 [μM] groups that attached to acetylated tubulin after M-100 treatment The chaperone DNAJA3 is recruited to acetylated tubulin and were HSPs from the chaperone type, including DNAJ proteins induces MYC degradation (Fig. 5F). For example, the chaperone member DNAJA3 occurred We verified the highly increased binding of DNAJA3 to acetylated which is important for proteasomal degradation and was shown tubulin after M-100 treatment using IP techniques and over- to interact with MYC in high-throughput studies [39]. Taken expression of DNAJA3-Flag (Fig. 6A, Supplemental Fig. 9A). In together, we demonstrate that MYC degradation is associated addition, we demonstrated the endogenous presence of acety- with changes in the interactome of heavily acetylated tubulin. lated tubulin and DNAJA3 complexes in Ramos cells (Fig. 6B, Oncogene (2022) 41:4560 – 4572 R. Winkler et al. 4566 Fig. 3 Different human B-cell lymphoma cell lines respond to HDAC6 inhibition by initiating apoptosis and cell cycle arrest. A Cell viability of human B-cell lymphoma cells after treatment with increasing concentrations of M-100 for 48 h using MTT assay. Non-linear regression (inhibitor vs. normalized response) was inserted. B Amount of Annexin V+ cells after treatment with 4 µM M-100 for 48 h. Unpaired Welch’s t-test, two-tailed. C Cell cycle analysis of cells treated with 4 µM M-100 for 48 h. Two-Way ANOVA (Sidak’s posthoc). D Different concentrations of M-100 were tested for inducing ac-H3 signals by Western blot. Vinculin was used as a loading control. E Ramos HDAC6 (HD6) knock-out (KO) cells were generated and compared to HDAC6 wild-type (WT) cells. Western Blot analysis shows absence of HDAC6. Actin serves as a loading control. F Proliferation was measured of Ramos HDAC6 WT and HDAC6 KO cells by cell counting, and normalized to WT cells at t = 72 h. Two-Way ANOVA (Sidak’s posthoc). G Dose-response curves were determined for Ramos HDAC6 WT and KO cells treated for 48 h with increasing concentrations of M-100 or ACY-1215 by MTT assay. Non-linear regression (inhibitor vs. normalized response) was inserted. Data in A–G are representative of at least n = 3 independent experiments. Data represent mean + SEM, if applicable. *P < 0.05, **P < 0.01, ***P < 0.001, ns not significant. Supplemental Fig. 9B). Of note, the chaperone DNAJA3 has large tubulin, which might involve other HSPs in the observed MYC and small isoforms that are generated from the cleavage of instability as well. precursor proteins [40, 41], which we could also detect in our The turn-over of MYC is depending on two opposing experiments (Fig. 6A, B). We used PLA to uncover a close cellular phosphorylation events of MYC at T58 and S62 which determines localization of DNAJA3 with MYC in B-cell lymphoma cells (Fig. 6C). protein stability as a phosphodegron [48, 49]. Our data show that However, HDAC6 inhibition rapidly reduced the number of Raji cells, which are mutated at T58, were the least sensitive to detected PLA foci per cell. These data suggest that tubulin acts M-100. Thus, efficient MYC degradation after HDAC6 inhibition as an interaction hub for MYC, HDAC6, and DNAJA3 in the might require wild-type T58 in MYC. Interestingly, phosphorylation cytoplasm, and switching the acetylation state of tubulin to of MYC impeded its interaction with tubulin, resulting in increased hyperacetylation results in disappearance of MYC. MYC stability in BL [48]. Conversely, our data indicate that the As DNAJ proteins are involved in ATP-dependent protein association of MYC with acetylated tubulin decreases MYC folding and degradation [42], we investigated potential effects stability. Moreover, our interactome data suggest that hyperace- of DNAJA3 on MYC stability. Surprisingly, both DNAJA3 isoforms tylation of tubulin leads to recruitment of proteins related to the were able to significantly decrease high MYC levels in over- functional annotation terms “Phosphoprotein”, “Nucleotide-bind- expression studies in a dose-dependent manner (Fig. 6D). We ing”, and “Ubiquitin-like conjugation” which may influence the retrospectively investigated the bone marrow of Eµ-Myc mice phosphodegron of MYC. It is likely that microtubule acetylation where MYC-overexpressing B-cells develop. When Eµ-Myc or wild- prolongs cytoplasmic retention of MYC making it accessible for type mice were treated once with 30 mg/kg M-100, we were able proteasomal degradation after interaction with HSPs, such as to detect elevated levels of Dnaja3 (Fig. 6E, Supplementary DNAJA3. Fig. 10). Increased levels of Dnaja3 after M-100 treatment might We did not discover direct effects of M-100 on the transcription further explain the absence of lymphomagenesis in Eµ-Myc mice. of the gene encoding MYC neither in murine nor in human cells. Taken together, our findings indicate that HDAC6 inhibition This observation is in contrast to the effects of the pan-HDACi MS- results in (1) a remodeling of the tubulin interactome driven by 275 in hematologic malignancies, which impaired transcription of hyperacetylation, (2) recruitment of HSPs including DNAJA3 to the MYC gene itself [13]. It was underlined by several studies that acetylated tubulin, and (3) degradation of excessive MYC in MYC- M-100 treatment does not result in altered histone acetylation overexpressing cells leading to apoptosis (Fig. 6F). Importantly, our [22, 50, 51]. results could be of beneficial use for the therapy of human MYC- The occupation of MYC on distinct sets of target genes was dependent lymphoid malignancies. described to depend on the amount of MYC molecules [5], which might explain the adverse Bcl-2 response of murine healthy and MYC-transformed B-cells to M-100 in our experiments. Eµ-Myc DISCUSSION lymphoma cells treated with M-100 de-repressed Bbc3 as well as The role of HDAC6 inhibitors in cancer therapy is still a matter of Pmaip1 and repressed Bcl2 expression. Importantly, direct debate. Recent studies showed that many cancer models are transcriptional regulation of Bbc3 and Pmaip1 by MYC was shown tolerant to HDAC6 inhibitor treatment [43–45]. However, a deeper to be possible [52, 53], and deficiency of Bbc3 and Pmaip1 look into the different tumor models considering newly emerging accelerated lymphomagenesis in Eµ-Myc mice [54]. Thus, perma- inhibitors might be helpful in developing new strategies for nent upregulation of Bbc3 and Pmaip1 caused by HDAC6 cancer treatment. Here, we demonstrated that MYC-dependent inhibition might be another explanation for the extended survival lymphomas are extremely sensitive to the highly specific HDAC6 of Eµ-Myc mice upon M-100 treatment. inhibitor M-100. Taken together, M-100 has potent anti-tumoral activity by In our work, we reveal that M-100 treatment of B-cell lymphoma targeting the stability of MYC. A fully water-soluble derivative of cells induces proteasomal degradation of MYC. Besides, we prove M-100 exists, which extends possible in vivo use [23]. However, that MYC forms a complex with the HSP DNAJA3, and drug resistance to HDAC6 inhibitors was described [55], and future hyperacetylation of tubulin strongly recruits DNAJA3. Studies directions should aim for rational drug combinations to treat showed that DNAJA3 associates directly with the E3 ligases distinct malignancies. HUWE1 and von Hippel-Lindau tumor suppressor [46]. HUWE1 is a major E3 ligase for MYC [47], which would connect DNAJA3 to the turnover of MYC. Besides, DNAJA3 was shown to mediate MATERIALS AND METHODS ubiquitination and degradation of oncogenic epidermal growth Please refer to supplementary information for standard biochemical or factor receptor [40]. Previous strategies to inhibit HDAC6 using molecular techniques. ACY-1215 in B-cell lymphoma resulted in an activation of the unfolded protein response by increasing quantity and acetylation In vivo animal studies of HSPs [18]. Similarly, we observed increased levels of the HSP Female and male C57BL/6JRj and Tg(IghMyc)22Bri (“Eµ-Myc”) mice with DNAJA3 in the bone marrow of mice treated with M-100. In C57BL/6JRj background were housed in individually ventilated cages in addition, our interactome analysis suggests that HDAC6 inhibition groups under specific-pathogen-free conditions in the Experimental leads to the recruitment of several chaperones to acetylated Biomedicine Unit at the University of Jena, Germany. Mice were bred Oncogene (2022) 41:4560 – 4572 R. Winkler et al. 4567 A 6h 24 h C 3h M-100 0 1 2 4 0 1 2 4 [μM] 70 kDa MYC 160 kDa 1.00 1.15 0.27 0.15 1.00 1.00 0.52 0.55 100 kDa Input Cl. PARP-1 Ub 100 kDa 25 kDa BCL-2 70 kDa 15 kDa 55 kDa Tub 55 kDa MYC Ramos Ub-MYC IP: α-MYC B Ub 4 μM M-100 0 6 6 3 3 0 [h] IgG 10 μM MG132 0 0 3 0 3 3 [h] MYC 70 kDa MYC MYC IgG 1.00 0.55 0.88 0.50 0.83 1.35 IP: IgG Ac-tub 55 kDa Ub GAPDH 35 kDa Ramos MYC IgG D Ramos 6h 24 h 6h 24 h 6h 24 h 6h 24 h 4 μM M-100 + + + + + + + + MYC Ac-tub Vinculin Raji BL-30 SUDHL-6 OCI-Ly3 Fig. 4 HDAC6 inhibition results in rapid MYC degradation. A Western Blot analysis of Ramos cells treated for the indicated time with different concentrations of M-100. Levels of MYC were quantified to untreated conditions. Tubulin was used as a loading control. Cl. - cleaved. B Western Blot analysis of Ramos cells treated with 10 µM MG132 to block proteasomal degradation and/or 4 µM M-100 for the indicated time. Levels of MYC were quantified to untreated conditions. GAPDH was used as a loading control. C Ubiquitination of proteins was analyzed in Ramos cells treated for 3 h with 10 µM MG132, 4 µM M-100 or left untreated by Western blot. 50 µg protein was loaded for input. Endogenous MYC was immunoprecipitated from these cell lysates. Unspecific IgG was used for control IPs. Ub - Ubiquitin. D Western Blot analysis of B-cell lymphoma cell lines treated for 6 h and 24 h with 4 µM M-100. Vinculin was used as a loading control. Ac-tub - acetylated tubulin. Data in A–D are representative of n = 3 independent experiments. according to registration number 02-053/16, and all experimental Activation of primary B-cells was induced by the addition of 10 µg/ml LPS procedures were approved by the federal Thuringian “Landesamt für (from E. coli, O111:B4, Sigma-Aldrich, Cat#L4391). Ramos cells were Verbraucherschutz” under registration number 02-030/15. All legal authenticated by Eurofins Genomics (Ebersberg, Germany) using PCR- specifications were followed regarding European guideline 2010/63/EU. single-locus-technology with 21 independent PCR-systems. M-100 was Mice were randomly assigned to different treatment groups and sacrificed dissolved to a 10 mM stock solution with DMSO and diluted to a by cervical dislocation or CO2 inhalation. For intraperitoneal injections, concentration of 100 µM with PBS. M-100 is registered under patent number M-100 was solved in 7.5% N-methyl-2-pyrrolidone (Carl Roth, Karlsruhe, WO2016020369 A1. Venetoclax (Selleck Chemicals, Houston, TX, United Germany, Cat#P052) and 40% PEG-400 (Carl-Roth, Cat#0144) in States) was solved in DMSO. sterile water. Flow cytometry Cell lines and primary cultures All measurements were performed using a LSR Fortessa system (BD All cell lines were maintained in incubators at 37 °C and 5% CO2. 293T and Biosciences, Franklin Lakes, NJ, United States), and data were acquired with NIH-3T3 cells were grown in DMEM with 10% FCS (Sigma-Aldrich, St. Louis, BD FACSDIVA V8.0.1 (BD Biosciences). Immune cell phenotyping was MO, United States, Cat#F7524). Ramos, Raji, and MV4-11 cells were grown in performed by staining single-cell suspensions in PBS with respective RPMI 1640 with 10% FCS. BL-30 cells were grown in RPMI 1640 with 20% FCS. antibodies. For apoptosis detection, an Annexin V-FITC Apoptosis OCI-Ly3, SUDHL-6, and CH12F3 cells were grown in RPMI 1640, supplemen- Detection Kit (Thermo Fisher Scientific, RRID:AB_2575600) was used. Cell ted with 10% FCS, 50 µM β-mercaptoethanol, 10 mM HEPES. CH12F3 cells cycle analysis was performed with fixed cells by propidium iodide (PI) were activated by stimulation with 1 µg/ml CD40L (Thermo Fisher Scientific, incorporation. Flow cytometry data were analyzed with FlowLogic 700.2A Waltham, MA, United States, Cat#16-0402-81, RRID:AB_468944), 5 ng/ml IL-4 (Inivai Technologies, Mentone, Australia). (Thermo Fisher Scientific, Cat#14-8041-62) and 1 ng/ml TGF-β1 (Cell Signaling Technology, Danvers, MA, United States, Cat#8915). All cell lines were tested regularly for Mycoplasma infection. Isolation of primary B-cells was performed Immunoprecipitation as previously described [56]. Primary B-cells were grown in RPMI 1640 with Protein levels were determined using the Roti-Nanoquant solution (Carl 10% FCS, 50 µM β-mercaptoethanol, 10 mM HEPES and 0.5% gentamicin. Roth GmbH, Cat#K880). For IPs, lysates containing 250 µg (overexpressed) Oncogene (2022) 41:4560 – 4572 R. Winkler et al. 4568 A B C Input MFI MYC-GFP (normalized) 4 μM M-100 + + + C N 1.0 HDAC6 HDAC6 MYC GFP 130 kDa Nucleus LMB 70 kDa MYC MYC IMP 0.5 MYC IgG MYC GFP HDAC1 Longer 55 kDa Cytoplasm exposure 0 Tubulin 55 kDa DMSO MeOH IMP LMB Ramos Ramos D CTR 4 μM M-100 CTR PLA: MYC + Ac-tub PLA: IgG 50 P<0.001 Foci number (mean per cell) 40 30 PLA + DAPI 20 10 0 CTR - - M-100 IgG E Ac-tub interactome F M-100 8 Keywords n adj. P Keyword: Chaperone Acetylation 130 6.4E-20 DNAJ proteins Phosphoprotein 204 1.8E-12 Other chaperones New binding 323 Nucleotide-binding 72 1.0E-10 6 None Protein transport 24 3.2E-3 Cytoplasm 108 3.2E-3 log2FC Upregulated binding 357 4 Keywords n adj. P (>2-fold) Acetylation 185 5.7E-44 Transit peptide 45 1.0E-14 Phosphoprotein 229 9.9E-13 2 Lost binding 108 Ubl conjugation 56 6.6E-4 Chaperone 14 1.3E-3 Fig. 5 Cytoplasmic MYC degradation is associated with changes in the interactome of hyperacetylated tubulin. A Interaction of MYC and HDAC6 detected by IP. Ramos cells were treated for 24 h with 4 µM M-100 or left untreated. IPs with unspecific IgG were used as control. B Cytoplasmic and nuclear fractions were prepared from Ramos cells. HDAC1 was used as a nuclear marker and tubulin as a cytoplasmic marker. C NIH-3T3 cells overexpressing MYC-GFP were challenged for 1 h with inhibitors of nuclear import (Importazole, IMP; 40 µM), nuclear export (Leptomycin B, LMB; 20 ng/ml), or solvent. Next, cells were treated for 90 min with CHX (50 µg/ml) before flow cytometry. Living cells were gated using FSC/SSC and normalized median fluorescence intensity (MFI) of MYC-GFP was calculated. Data represent mean + SEM. Unpaired Student’s t-test, two-tailed. D PLA was performed to detect endogenous co-localization of MYC and acetylated tubulin (ac-tub) in Ramos cells. Cells were treated with 4 µM M-100 for 24 h. Staining with unspecific IgG was used as a control. DAPI was used to stain nuclei. Scale bars indicate 20 µm, magnification 60x. PLA foci were counted and compared. Boxplots depict medium and min to max. One-Way ANOVA (Tukey’s posthoc). E Global interactome analysis was carried out of immunoprecipitated ac-tub via mass spectrometry. MV4-11 cells were treated for 24 h with 0.5 µM M-100. Shown are counts of proteins with new or increased (>2-fold) binding to ac-tub, or loss of binding after treatment compared to control and IgG binding. Uniprot (UP) keyword annotation was performed with DAVID using all proteins that bound to ac-tub after M-100 treatment. Adjusted (adj.) P-values are given. Ubl - Ubiquitin-like. F All proteins belonging to the keyword “chaperone” are depicted with their corresponding log2 fold change (FC). DNAJ proteins are marked in green. Data in A and C are representative of n = 3 independent experiments. Data in B and D are representative of n = 2 independent experiments. Oncogene (2022) 41:4560 – 4572 R. Winkler et al. 4569 A B Input IP: α-Flag Input IP: α-DNAJA3 IP: IgG 1 μM M-100 + + + + M-100 0.5 4 0.5 4 0.5 [μM] DNAJA3-Flag + + + + MS-275 5 5 [μM] Ac-K Ac-tub Ac-K Ac-tub IgG DNAJA3 DNAJA3 (up) DNAJA3 (L) DNAJA3 DNAJA3 DNAJA3 (S) IgG 293T Ramos C CTR 4 μM M-100, 3 h 4 μM M-100, 24 h CTR PLA: MYC + DNAJA3 PLA: IgG P=0.003 30 Foci number (mean per cell) P=0.020 20 PLA + DAPI 10 0 0 h 3 h 24 h IgG D E + MYC + MYC Eμ-Myc mice 100 DNAJA3 (S) + ++ DNAJA3 (L) + ++ FC Dnaja3 Vehicle + + MYC MYC M-100 + + 55 DNAJA3 (up) DNAJA3 DNAJA3 Dnaja3 Dnaja3 (L) DNAJA3 Dnaja3 (S) 00 130 kDa Vinculin Vinculin Vinculin 100 kDa 293T 293T Bone marrow F MYC 1.0 1.0 E-Box Gene MYC (normalized) MYC (normalized) MYC Nucleus HDAC6 Cytoplasm Ac 0.5 0.5 Ac Chaperones DNAJ MYC Degradation 0.0 0.0 DNAJA3 + ++ DNAJA3 + ++ Hyperacetylation (+ M-100) or 1000 µg (endogenous) protein were combined with a mixture of 50% (v/ Proximity ligation assay v) protein A and 50% (v/v) protein G beads (Sigma-Aldrich Inc., Cat#P9424 PLA was performed using Duolink In Situ PLA Reagents Red (Sigma-Aldrich, and Cat#P3296) and 0.5-1 µg antibody in lysis buffer overnight at 4 °C. Cat#DUO92008) according to the manufacturer’s protocol. Ramos cells Following control antibodies were used: Mouse IgG control (Santa Cruz were attached to coverslips with 0.1% poly-L-lysine (Sigma-Aldrich, Biotechnology, Dallas, TX, United States, Cat#sc-2025, RRID:AB_737182), Cat#P8920) for 1 h at RT. Cells were permeabilized, fixed with methanol Rabbit IgG control (Santa Cruz Biotechnology, Cat#sc-2027, RRI- for 10 min at −20 °C, blocked with Duolink blocking solution, and D:AB_737197). Beads were washed three times in lysis buffer after incubated with primary antibodies. PLA Probe Anti-Mouse MINUS incubation, resuspended in 2x Laemmli buffer to a final 1x concentration, (Sigma-Aldrich) and PLA Probe Anti-Rabbit PLUS (Sigma-Aldrich) were and boiled for 5 min at 95 °C. used as secondary probes. Samples were mounted with DAPI. PLA signals Oncogene (2022) 41:4560 – 4572 R. Winkler et al. 4570 Fig. 6 The heat-shock protein DNAJA3 is recruited to hyperacetylated tubulin and induces MYC degradation. A Interaction of acetylated tubulin (ac-tub) and DNAJA3. 293T cells were transfected with plasmids encoding DNAJA3-Flag and treated with 1 µM M-100 for 24 h. Cells were lysed in stringent lysis buffer containing M-100. Lysates were used for IP with α-Flag antibodies to precipitate DNAJA3-Flag. Overexpression of DNAJA3 generates unprocessed (up) precursor proteins. B Endogenous interaction of acetylated tubulin and DNAJA3 in Ramos cells. Cells were treated for 24 h with either 0.5 µM, 4 µM M-100, 5 µM MS-275, or left untreated. Lysates were used for IP with α-DNAJA3 antibodies and tested for interaction with ac-tub. IPs with unspecific IgG were used as control. C PLA was performed to detect endogenous co- localization of MYC and DNAJA3 in Ramos cells. Cells were treated with 4 µM M-100 for the indicated time points. Staining with unspecific IgG was used as a control. DAPI was used to stain nuclei. Scale bars indicate 20 µm, magnification 60x. PLA foci were counted and compared. Boxplots depict medium and min to max. One-Way ANOVA (Tukey’s posthoc). D Western Blot analysis of 293T cells overexpressing MYC and increasing amounts of small (S) or large (L) isoforms of DNAJA3. Vinculin was used as a loading control. Quantification of MYC was performed based on Vinculin. One-Way ANOVA (Dunnett’s posthoc). E Western Blot analysis of bone marrow lysates from Eµ-Myc mice after one i.p. injection with M-100 (30 mg/kg) or vehicle. Small and large isoforms of Dnaja3 can be noticed. Vinculin was used as a loading control. Each lane represents one individual mouse. Quantification of Dnaja3 protein levels is shown based on Vinculin. FC - fold change. F Scheme summarizing our findings. Hyperacetylation of tubulin by HDAC6 inhibition results in the recruitment of chaperone complexes including the HSP DNAJA3. 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Lechner S, Malgapo MIP, Grätz C, Steimbach RR, Baron A, Rüther P, et al. Target Glowalla, Petra Grübner, and Andreas Köber for animal care. We are grateful to Katrin deconvolution of HDAC pharmacopoeia reveals MBLAC2 as common off-target. Schubert for assistance with cell sorting (FLI Jena) and Dr. David Corujo for Nat Chem Biol. 2022. bioinformatic support. We also want to thank Dr. Marcus Buschbeck and Dr. Jeannine 39. Heidelberger JB, Voigt A, Borisova ME, Petrosino G, Ruf S, Wagner SA, et al. Diesch for giving feedback on the manuscript. Prof. Dr. Ralf Küppers kindly provided Proteomic profiling of VCP substrates links VCP to K6‐linked ubiquitylation and c‐ lymphoma cell lines. Venetoclax was a kind gift from Prof. Dr. Sebastian Scholl and Dr. Myc function. EMBO Rep. 2018;19:1–20. Maximilian Fleischmann. 40. Chen CY, Jan CI, Lo JF, Yang SC, Chang YL, Pan SH, et al. Tid1-L inhibits EGFR signaling in lung adenocarcinoma by enhancing EGFR ubiquitinylation and degradation. Cancer Res. 2013;73:4009–19. 41. Lu B, Garrido N, Spelbrink JN, Suzuki CK. Tid1 isoforms are mitochondrial DnaJ- AUTHOR CONTRIBUTIONS like chaperones with unique carboxyl termini that determine cytosolic fate. J Biol Conception and design, CK; Development of methodology, RW, A-SM, MB, MEH, SP, Chem. 2006;281:13150–8. and FH; Acquisition of data, RW, A-SM, E-MP, MK, MB, KL, LH, A-MS, MEH, SP, and FH; 42. Sterrenberg JN, Blatch GL, Edkins AL. Human DNAJ in cancer and stem cells. Analysis and interpretation of data, RW, A-SM, E-MP, MEH, and CK; Writing, review, Cancer Lett. 2011;312:129–42. and/or revision of the manuscript, RW, A-SM, and CK; Administrative, technical, or 43. Depetter Y, Geurs S, De Vreese R, Goethals S, Vandoorn E, Laevens A, et al. material support, OHK, PB, and SM; Study supervision, PB, TM, OHK, and CK. Selective pharmacological inhibitors of HDAC6 reveal biochemical activity but functional tolerance in cancer models. Int J Cancer. 2019;145:735–47. 44. Wachholz V, Mustafa AHM, Zeyn Y, Henninger SJ, Beyer M, Dzulko M, et al. Inhibitors of class I HDACs and of FLT3 combine synergistically against leukemia FUNDING cells with mutant FLT3. Arch Toxicol. 2022;96:177–93. RW was supported by a scholarship from the Carl Zeiss Foundation. CK received 45. Beyer M, Romanski A, Mustafa AHM, Pons M, Büchler I, Vogel A, et al. Hdac3 funding from the Deutsche Forschungsgemeinschaft (DFG, German Research activity is essential for human leukemic cell growth and the expression of Foundation), GRK 1715. E-MP was supported by a Landesgraduiertenstipendium catenin, myc, and wt1. Cancers 2019;11:1–20. (Friedrich Schiller University Jena). Work done in the group of OHK was done by MB 46. Thompson JW, Nagel J, Hoving S, Gerrits B, Bauer A, Thomas JR, et al. Quantitative and is funded by DFG, Project-ID 393547839–SFB 1361 and DFG, GRK 2291/9-1, Lys-∈-Gly-Gly (diGly) proteomics coupled with inducible RNAi reveals ubiquitin- project number 427404172. Open Access funding enabled and organized by mediated proteolysis of DNA damage-inducible transcript 4 (DDIT4) by the E3 Projekt DEAL. Ligase HUWE1. J Biol Chem. 2014;289:28942–55. 47. Inoue S, Hao Z, Elia AJ, Cescon D, Zhou L, Silvester J, et al. Mule/Huwe1/Arf- BP1 suppresses Ras-driven tumorigenesis by preventing c-Myc/Miz1-mediated down-regulation of p21 and p15. Genes Dev. 2013;27:1101–14. COMPETING INTERESTS 48. Niklinski J, Claassen G, Meyers C, Gregory MA, Allegra CJ, Kaye FJ, et al. Disruption The authors declare no competing interests. of Myc-tubulin interaction by hyperphosphorylation of c-Myc during mitosis or by constitutive hyperphosphorylation of mutant c-Myc in Burkitt’s lymphoma. Mol Cell Biol. 2000;20:5276–84. ADDITIONAL INFORMATION 49. Popov N, Schülein C, Jaenicke LA, Eilers M. Ubiquitylation of the amino terminus Supplementary information The online version contains supplementary material of Myc by SCFβ-TrCP antagonizes SCFFbw7-mediated turnover. Nat Cell Biol. available at https://doi.org/10.1038/s41388-022-02450-3. 2010;12:973–81. 50. Schäfer C, Göder A, Beyer M, Kiweler N, Mahendrarajah N, Rauch A, et al. Class I Correspondence and requests for materials should be addressed to Christian Kosan. histone deacetylases regulate p53/NF-κB crosstalk in cancer cells. Cell Signal. 2017;29:218–25. Reprints and permission information is available at http://www.nature.com/ 51. Stojanovic N, Hassan Z, Wirth M, Wenzel P, Beyer M, Schäfer C, et al. HDAC1 and reprints HDAC2 integrate the expression of p53 mutants in pancreatic cancer. Oncogene 2017;36:1804–15. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims 52. Wirth M, Stojanovic N, Christian J, Paul MC, Stauber RH, Schmid RM, et al. MYC in published maps and institutional affiliations. and EGR1 synergize to trigger tumor cell death by controlling NOXA and BIM Oncogene (2022) 41:4560 – 4572 R. Winkler et al. 4572 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http:// creativecommons.org/licenses/by/4.0/. © The Author(s) 2022 Oncogene (2022) 41:4560 – 4572