Negative feedback regulation of MAPK signaling is an important driver of CLL progression

doi:10.21203/rs.3.rs-2481423/v1

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Negative feedback regulation of MAPK signaling is an important driver of CLL progression

https://doi.org/10.21203/rs.3.rs-2481423/v1

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Despite several potent targeted treatments for chronic lymphocytic leukemia (CLL), the clinical challenge of treating drug-resistant disease is emerging. In this study, we discovered that the dual-specific phosphatases DUSP1 and DUSP6 are required to negatively regulate Mitogen-activated protein kinases (MAPK) and thus counterbalance excessive MAPK activity to prevent apoptosis in CLL. We show that DUSP1 and DUSP6 are widely expressed in CLL and high expression of DUSP6 in CLL correlates with a poor clinical prognosis, which may reflect high levels of MAPK activity. Importantly, genetic deletion of the inhibitory phosphatase DUSP1 or DUSP6 and blocking DUSP1/6 function using a small molecule are toxic for CLL cells in vitro and in vivo. Analyzing downstream effects using global phospho-proteome approaches, we observed that acute activation of MAPK signaling by DUSP1/6 inhibition induces DNA damage response and thereby apoptotic cell death in CLL cells. This cell death is mediated by CHK kinases and can function independent of p53 and ATM, both effectors of DNA damage response, which are frequently deleted in CLL. Finally, we observed that DUSP1/6 inhibition is particularly effective against treatment-resistant CLL and therefore suggest transient DUSP1/6 inhibition as a promising novel treatment concept to eliminate drug-resistant CLL cells.

Biological sciences/Cancer/Haematological cancer/Leukaemia/Chronic lymphocytic leukaemia

Biological sciences/Cell biology/Cell signalling

Health sciences/Diseases/Cancer/Cancer therapy/Targeted therapies

Biological sciences/Cell biology/Cell death/Apoptosis

Biological sciences/Cancer/Cancer therapy/Targeted therapies

Chronic lymphocytic leukemia (CLL) is among the most frequent B-cell malignancies in the Western world and is characterized by clonal accumulation of mature CD5-positive B-cells. Signaling from the B-cell receptor (BCR) plays a significant role in promoting CLL pathogenesis.^1–4 Inhibiting BCR downstream effectors with the BTK inhibitor ibrutinib, among others, is highly effective in lowering the disease burden and has transformed CLL therapy in recent years. Despite these targeted therapies, CLL remains an incurable disease for most patients, and the emergence of drug-resistant CLL demands the exploration of alternative treatments.⁵

Activation of the mitogen-activated protein kinase (MAPK) pathway promotes CLL cell proliferation and survival and occurs downstream of surface receptors, including the BCR. In addition, the MAPK-signaling pathway is frequently activated by mutations in CLL, with 8.7% of patients carrying at least one mutation in MAPK-pathway genes such as NRAS, KRAS, and BRAF.⁶ Importantly, patients with MAPK-activating mutations in CLL comprise a distinct subgroup with poor clinical characteristics such as shorter treatment-free survival^{7, 8} and higher drug resistance ^9–11. However, clinical studies of FDA-approved MEK-or BRAF-inhibitors for CLL have yielded poor outcomes.¹² As a result, despite its importance in CLL etiology, elevated MAPK signaling is yet to be addressed therapeutically.

The physiological outcome of MAPK signalling in B-cells varies greatly depending on the maturation stage and activation status.¹³ ERK1/2 activation, for example, is predominantly known for its pro-survival effects. However, ERK1/2 activation can trigger apoptotic processes in developing B-cells, which are important for eliminating autoreactive B-cells.^{14, 15} Thus, the actual role of MAPK activation is highly cell- and context-dependent. MAPK is negatively regulated by the expression of dual-specificity protein phosphatases (DUSPs), which specifically dephosphorylate phosphothreonine and -tyrosine residues that activate MAPKs. Two distinct members of the DUSP family, DUSP1 and DUSP6, inactivate ERK1/2 by dephosphorylation. Although DUSP6 is specific for ERK1/2, DUSP1 also dephosphorylates JNK and p38. Both DUSPs are dysregulated in various diseases, including cancer. They can either function as tumor suppressors by inhibiting ERK activation^{16, 17} or as oncogenes by assisting tumor cells in adapting to high levels of MAPK signaling^{18, 19}. Although adaption to signaling levels is critical for a number of B-cell malignancies,¹⁹ the role of MAPK negative feedback via the DUSP family in CLL remains largely unknown.

In the present study, we explored the importance of DUSP1- and DUSP6-mediated negative regulation of MAPK in CLL and identified DUSP6 overexpression as a marker of enhanced MAPK activity, and hence, poor clinical prognosis in CLL. We showed that DUSP1 and DUSP6 are necessary in CLL to avoid apoptosis via the DNA damage response pathway by counterbalancing excessive oncogenic MAPK activation. Therefore, the inhibition of DUSP1 and DUSP6 significantly reduced the CLL burden in preclinical models and was highly effective in the treatment of drug-resistant CLL.

Expression of the MAPK negative regulator DUSP6 but not DUSP1 is associated with poor clinical prognosis in CLL and may reflect ERK activity.

To investigate the potential role of negative feedback regulation of MAPK signaling in CLL, we first analyzed the mRNA expression levels of DUSP1 and DUSP6 in a total number of 210 CLL samples.²⁰ We observed that both DUSP1 and DUSP6 were readily expressed in all tested CLL samples, with higher levels of DUSP6 and lower levels of DUSP1 in CLL cases carrying unmutated BCRs, which is one of the strongest predictive factors for poor disease outcome (Fig. 1a, b). We also evaluated whether DUSP1 and DUSP6 expression is associated with CLL prognosis. We correlated the mRNA expression levels of DUSP1 and DUSP6 with time to treatment (TTT) and overall survival (OS).^{20, 21} DUSP1 expression was not associated with clinical parameters (Suppl. Figure 1a, b), we found that high expression of DUSP6 in CLL samples was associated with adverse clinical outcomes, indicated by decreased TTT (Fig. 1c) and OS (Fig. 1d). Analysis of DUSP6 expression in the mutated or unmutated CLL subsets indicated that high DUSP6 levels are only significantly associated with shorter TTT in the mutated CLL subset, which could be due to the limited number of patients analyzed (Suppl. Figure 1c). However, we noticed that CLL samples with mutations in the KRAS and BRAF genes (causing high levels of MAPK signaling) expressed very high levels of DUSP6 (Fig. 1e). However, this was not the case when we compared DUSP1 levels in CLL samples with KRAS and BRAF mutations (Suppl. Figure 1d). As negative regulators are commonly induced in response to stimulation,^{22, 23} we hypothesized that the levels of DUSP6 in CLL cells are directly related to the degree of MAPK signaling ²⁴. To test whether MAPK signaling affects DUSP6 expression levels, we analyzed the effects of inhibition of MAPK signaling by the small molecule inhibitor PD0325901 (PD901) on DUSP6 levels and observed a significant reduction in DUSP6 in conjunction with reduced ERK phosphorylation (Fig. 1f, g). Stimulation of CLL cells by microenvironmental factors engages the BCR and MAPK signaling pathways in CLL cells, so we also analyzed DUSP6 expression in matched samples collected from the peripheral blood and lymph nodes using publicly available data.²⁵ Indeed, DUSP6 and DUSP1 expression was higher in lymph node-derived CLL cells than in the respective peripheral blood CLL samples (Fig. 1h-i). Taken together, we conclude that the MAPK-negative regulators DUSP1 and DUSP6 are commonly expressed in CLL and are dynamically upregulated in response to microenvironmental stimulation. High levels of DUSP6 expression, but not DUSP1 expression, define a subset of patients with poor prognosis, particularly those with KRAS and BRAF mutations.

DUSP1/6 inhibition is toxic specifically for CLL cells

To investigate the functional relevance of DUSP1- and DUSP6-mediated negative MAPK regulation in CLL, we tested the effects of the DUSP1/6- specific small molecule inhibitor BCI²⁶ on CLL cells. We treated 21 primary CLL samples harvested from the peripheral blood of patients for 48h with increasing doses of BCI and found a dose-dependent induction of specific cell death in vitro (Fig. 2a). We calculated the specific cell death in vehicle-treated control CLL cells undergoing spontaneous apoptosis in cell culture.²⁷ There was no significant difference in the response of CLL to BCI based on the mutational status of their BCR (Suppl. Figure 1e). In addition, cell death was specifically induced in CLL cells, whereas B-cells derived from healthy donors remained largely unaffected by DUSP1/6 inhibition at the tested concentrations (Fig. 2b). Furthermore, we confirmed the selective toxicity of the DUSP1/6 inhibitor to CLL using the CLL-derived cell line MEC-1 compared to most other tested B-cell and T-cell lymphoma cell lines (Fig. 2c).

To test whether the pharmacological inactivation of DUSP1/6 is a potential therapeutic option for CLL, we also determined its effect on CLL progression in vivo, using a TCL1-driven mouse model.²⁸ For in vivo treatments, we used a derivate of BCI (BCI-215) with reduced in vivo toxicity.^29,30 We injected CLL-bearing splenocytes from aged TCL1-tg mice into WT recipients and confirmed CLL engraftment prior to treatment initiation (Fig. 2d). Then the mice were randomized to treatment or control groups and were treated with either BCI-215 (10 mg/kg) or vehicle daily for 10 consecutive days. This resulted in significantly reduced CLL content in the spleen and the peritoneal cavity, the major target organs for TCL1-derived murine CLL,²⁸ in the BCI215 treated mice as compared to the control mice (Fig. 2e, f). Taken together, we showed that CLL cells are highly sensitive to DUSP1/6 inhibition, suggesting that negative regulation of the MAPK signaling pathway is important for their survival and disease progression in vitro and in vivo.

Genetic disruption of DUSP1 or DUSP6 impairs CLL cell expansion

To exclude the possibility that the cytotoxic effects of BCI observed in CLL are primarily due to off-target effects by the small-molecule inhibitor, we next performed genetic knockout experiments to determine the role of DUSP1 and DUSP6 in CLL cell survival. To this end, we generated CRISPR/Cas9-mediated knockout lines of CLL-derived MEC-1 cells. Upon successful gene knockout of DUSP1 or DUSP6 in expanded single clones, as verified by western blotting (Fig. 2g, h), we performed in vitro competitor growth assays to compare growth behaviors. For this, we mixed the expanded GFP-expressing clones (carrying either the DUSP1 or DUSP6 knockout or the CAS9 control vector) with untransduced MEC-1 cells to visualize changes in their growth behavior under similar growth conditions. While the respective Cas9/GFP + control cells were not affected (Fig. 2i, j), both DUSP1 and DUSP6 knockout clones were outcompeted by WT MEC-1 cells, confirming the selective disadvantage of CLL cells lacking functional DUSP1 or DUSP6. Our attempts to generate DUSP1/DUSP6 double-knockout MEC-1 lines failed, suggesting that these are lethal (not shown). Notably, DUSP1 and DUSP6 knockout cells showed a markedly diminished response to the DUSP1/6 inhibitor, indicating that the observed specific apoptosis was largely attributed to on-target effects (Fig. 2k). It should be noted that DUSP1 and DUSP6 knockout and CAS9 + control MEC-1 cells were expanded from single cells prior to these experiments, which may limit the observed effects and the reproducibility of the effects of acute DUSP1/6 inhibition. Nevertheless, these experiments confirm that the expression and activity of DUSP1 and DUSP6 are required for optimal CLL cell growth and survival.

DUSP1/6 inhibition induces BCR/MAPK signaling in CLL cells followed by DNA damage response and apoptosis

To assess DUSP1/6-mediated signaling events in CLL, we used an unbiased screening approach and performed global phospho-proteome analysis of BCI- or vehicle-treated CLL cells. We first analyzed the early time points of BCI treatment for total phospho-proteomic alterations to validate the on-target effects of DUSP1/6 inhibition and to study the initial events using patient-derived CLL cells (workflow depicted in Fig. 3a). After 10 min of DUSP1/6 inhibitor treatment, we observed significant changes in the phosphorylation profile (Fig. 3b; Suppl. Figure 2a, b). Phospho-proteomic pathway analysis revealed that the phospho-sites of the BCR and MAPK networks were significantly deregulated (Fig. 3c). As expected, we observed an increase in the phosphorylation of both ERK1 and ERK2 in DUSP1/6 inhibitor-treated CLL cells compared to control cells, confirming the on-target specificity of the inhibitor. Using western blotting, we validated the early phosphorylation of ERK1/2 upon treatment with the DUSP1/6 inhibitor in three additional primary CLL samples (Suppl. Figure 2c).

To account for the heterogeneity among individual CLL patients in the signaling responses induced by DUSP1/6 inhibition, we used the MEC-1 cell line as a model to study the downstream signaling events of DUSP1/6 inhibition in CLL. Here, we performed a time-course experiment with subsequent phospho-proteome analysis using MEC-1 CLL cells, using later time points (0, 15, and 45 min) to gain insight into the molecular events associated with cell death. A heatmap with differentially phosphorylated proteins over all analyzed time points is shown, with four different clusters identified (Fig. 4a, b). We then performed kinase prediction analysis to identify the kinases that mediate the observed downstream effects (Fig. 4c). We identified the target sites of several kinases, including CDK1, PKCβ, and CK2α. As expected, ERK1/2 target sites were significantly phosphorylated, confirming ERK1/2 activation upon DUSP1/6 inhibition. However, when we analyzed which kinases were most active upon DUSP1/6 inhibition, we observed that HIPK2-regulated target sites were highly phosphorylated upon DUSP1/6 inhibitor treatment at both time points (Fig. 4d; analysis for the remaining kinases depicted in Suppl. Figure 2d, e). HIPK2 is a serine/threonine-protein kinase involved in p53/TP53-mediated cellular apoptosis and the DNA damage response (DDR) pathway³¹ and is a downstream target of the MAPK pathway.³² Consistent with this, subsequent pathway analysis revealed that the DDR pathway is one of the most strongly regulated pathways upon DUSP1/6 inhibitor treatment in MEC-1 cells, with activating phosphorylation events on ATF2, c-JUN, and CHK1/2 kinases (Fig. 4e).^33,34 We validated the activation of these pathways following DUSP1/6 inhibition in primary CLL samples (Suppl. Figure 2f, g). In addition, we observed differential phosphorylation of molecules associated with the apoptotic pathway upon DUSP1/6 inhibition in our phospho-proteome screen, which is in line with the observed cell death upon DUSP1/6 inhibition in CLL (Fig. 4f). Taken together, our phospho-proteome analysis suggests that DUSP1/6 inhibition induces MAPK signaling, followed by activation of the DDR and apoptotic pathways in CLL.

Functional relevance of downstream signaling mediated by DUSP1/6 inhibition in CLL

Our phospho-proteome and western blot analyses revealed enhanced activation of the MAPK signaling pathway upon DUSP1/6 inhibition. To determine whether MAPK activation is associated with cell death induced by DUSP1/6 inhibition in CLL, we evaluated whether preventing ERK1/2 activation would mitigate the apoptotic effects of DUSP1/6 inhibition. Although MEK inhibition itself is toxic to CLL cells,³⁵ we observed that co-treatment with the MEK inhibitor PD901 partially reversed the induction of specific cell death by the DUSP1/6 inhibitor (Fig. 5a), indicating that ERK activation contributes to DUSP1/6-mediated cell death. Based on the activation of the DDR pathway observed in our phospho-proteome screen, we investigated whether DUSP1/6 inhibition promotes DNA damage in CLL cells. To this end, we analyzed the phosphorylation of γH2AX, one of the earliest cellular responses to DNA double-strand breaks, using flow cytometry. Indeed, phospho-γH2AX levels increased upon DUSP1/6 inhibitor treatment as compared to the control patient-derived CLL cells in a dose-dependent manner (Fig. 5b), while isotype control-stained cells did not show increased fluorescence (Suppl. Figure 3a). It should be noted that γH2AX phosphorylation occurs only after 1–4 hours of DUSP1/6 inhibitor treatment and was therefore not detected in our global phospho-proteome analysis, where we used early time points to avoid secondary effects on high levels of cell death. To determine whether the DNA damage response was a consequence of increased MAPK signaling induced by DUSP1/6 inhibition, we analyzed the phosphorylation of γH2AX with and without pretreatment with the MEK-inhibitor PD901 in MEC1, OSU-CLL, and EHEB CLL cell lines. We observed that PD901 treatment significantly reduced the DUSP1/6-mediated upregulation of phospho-γH2AX in multiple independent experiments (Fig. 5c).

In addition, we analyzed whether DUSP1/6 inhibition induces classical apoptosis in CLL, as indicated by phospho-proteome analysis. Annexin V staining of primary human CLL cells treated with DUSP1/6 inhibitor revealed strong exposure of phosphatidylserine on the outer membrane (Fig. 5d). Consistent with this, co-treatment with the pan-caspase inhibitor QVD (or Emericasan) significantly reduced the toxic effect of DUSP1/6 inhibition alone (Fig. 5e, Suppl. Figure 3b). Absolute CLL cell viability increased upon caspase inhibition by reducing spontaneous apoptosis in cultured CLL cells (Suppl. Figure 3c). Next, we investigated whether the observed activation of the DNA damage response pathway promoted the cytotoxic effects of DUSP1/6 inhibition in CLL cells. Therefore, we investigated whether inhibition of CHK1/2 kinases, critical effectors of the DDR pathway (which were also differentially phosphorylated in our MEC-1 phospho-proteome screen) reduced the cytotoxic effects of DUSP1/6 inhibition. CHK1/2 inhibition alone was not toxic to the CLL cells at the tested concentrations (Suppl. Figure 3d), concomitant treatment of CLL with DUSP1/6 and CHK1/2 inhibitors ameliorated the toxic effect of DUSP1/6 inhibition alone (Fig. 5f). The CHK1 inhibitor similarly reduced cell death induced by DUSP1/6 inhibition (Suppl. Figure 3e). Nevertheless, ATM inhibition did not significantly alter the effects of DUSP1/6 inhibition on CLL cell survival, indicating that this pathway is less important in the downstream response (Suppl. Figure 3f). In addition, p53, a major effector in the induction of apoptosis as a consequence of DNA damage, seems to be dispensable for mediating DUSP1/6 inhibition-promoted cell death, as the MEC-1 CLL cell line, which carries both 17p deletion (Del17p) and TP53 mutations, is sensitive to DUSP1/6 inhibition (shown in Fig. 2c). Taken together, our analysis confirmed the functional relevance of the DDR pathway in apoptosis induction in CLL cells upon DUSP1/6 inhibition, which is at least partially mediated by the activation of CHK1/2 and functions in the absence of ATM or p53 activity.

DUSP1/6 inhibition is effective in drug resistant CLL

Similar to the MEC-1 cell line, primary CLL cells frequently harbor genetic alterations in the DDR pathway. Up to 8% of chemotherapy-naïve patients carry Del17p, and up to 80% of these carry mutations in TP53 on the second allele.^{36, 37} This loss of functional p53 is associated with resistance to chemotherapeutic agents. In addition, ATM is frequently inactivated in CLL and is associated with reduced apoptosis induction in response to chemotherapeutic agents.³⁸ To determine whether genetic alterations in the DDR pathway affect the response rate to DUSP1/6 inhibition in CLL, we compared the cytotoxic response towards DUSP1/6 inhibition in CLL samples carrying p53 mutations or Del17p or Del11q, leading to a loss of functional p53 or ATM, respectively, as compared to WT p53 or ATM expressing cases. Although there was a minor reduction in the mean cell death induction, there was no significant difference in the cytotoxic response to DUSP1/6 inhibition between cases with and without functional p53/ATM (Fig. 5g). Although this analysis is based on a limited number of samples with the respective genetic alterations, it indicates that DUSP1/6 inhibition remains highly effective in killing CLL cells carrying mutations that disrupt a functional ATM/p53-mediated DDR.

Based on the clinical need to identify novel treatment options for clinically ibrutinib-refractory CLL, we compared the effects of DUSP1/6 inhibition on primary CLL in treatment-naïve patients with ibrutinib-refractory cases. Here, we found that ibrutinib-resistant CLL cells remained sensitive to BCI treatment with similar induction of specific cell death as compared to treatment-naïve CLL cells analyzed in parallel (Fig. 5h). Taken together, we showed that DUSP1/6 inhibition is highly effective in inducing cytotoxicity in all tested CLL subsets and may be particularly useful for treating treatment-resistant and refractory CLL.

In this study, we discovered a previously unknown role for DUSP1 and DUSP6 in limiting MAPK signaling in CLL, thereby preventing apoptosis. We discovered that high mRNA expression of the negative regulator DUSP6 is linked to a poor clinical outcome in CLL. Both DUSP1 and DUSP6 expression are necessary for maintaining optimal CLL cell proliferation. Inhibition of DUSP1 and DUSP6 phosphatase activities limits CLL cell survival in vitro and disease development in vivo. Mechanistically, we discovered that inhibiting DUSP1/6 triggers activation of the BCR/MAPK signaling pathway, causing DNA damage-mediated apoptosis. This pro-apoptotic effect of DUSP1/6 inhibition was strong in drug-resistant CLL. Therefore, we identified the MAPK negative regulators DUSP1 and DUSP6 as novel treatment targets, particularly for drug-resistant CLL.

By acting as negative regulators of the MAPK signaling pathway, dual-specific phosphatases can restrict tumor growth and promote carcinogenesis and drug resistance.³⁹ Understanding the downstream processes of DUSP1/6 inhibition should aid in determining malignancies that are responsive to the drug. We used an unbiased phospho-proteome screen to determine the cause of cell death in response to DUSP1/6 inhibition in CLL. ERK1/2 activation was found to be important for inducing CLL apoptosis upon DUSP1/6 inhibition, which may appear surprising as ERK1/2 activity is most commonly associated with cell proliferation and survival. However, active ERK1/2 has also been linked to cell death during negative selection of B-cells to prevent autoimmunity.¹⁴ We hypothesized that CLL-specific checkpoints that limit B-cell growth stem from the inherent sensitivity of B-cells to strong BCR/MAPK signaling that mimics autoimmunity, resulting in the indolent nature of the disease. Consequently, CLL cells die rather than proliferate rapidly when MAPK signaling is induced to physiological levels. In contrast to DUSP6, DUSP1 mRNA levels do not correlate with disease progression, which could be due to the regulation of DUSP1 at the post-transcriptional level.⁴⁰ Interestingly, Richter’s transformation is associated with an increased mutation frequency in the MAPK signaling pathway and co-occurring alterations in genes involved in the DNA damage response pathway.⁴¹ For example, loss of CHK1 and ATR was frequently observed in Richter transformed cases, but not in indolent CLL.⁴¹ We found that both molecules were strongly activated by DUSP1/6 inhibition and potential promoters of DUSP1/6 inhibition-mediated cell death in CLL. This suggests that growth-limiting checkpoints are lost, for example, by deletion of important DNA damage response mediators, as CLL transforms into aggressive lymphoma, where robust and sustained signaling is well tolerated. Therefore, the vulnerability of CLL cells to DUSP1/6 inhibition is likely characterized by effective CHK- and/or ATR-mediated apoptosis in response to MAPK hyperactivation. Further understanding of the biology of Richter’s transformation will help clarify this.

Drug resistance is a major obstacle to the treatment of CLL. Despite high initial response rates, a considerable percentage of patients receiving chemotherapy or targeted therapies experience relapse with progressive and refractory disease. Drug resistance has been linked to the mutation of specific drug-binding sites or the activation of alternative pro-survival pathways, including the MAPK signaling pathway.^9–11 In addition, particular genetic abnormalities are associated with drug resistance to chemotherapeutic treatments, such as the deletion of ATM or p53, which are important molecules in DNA damage-mediated apoptosis ^{36, 38}. Although we observed that the DNA damage response was also involved in DUSP1/6 inhibitor-mediated cytotoxicity, DUSP1/6 suppression efficiently killed p53-mutated and ATM-deleted CLL, suggesting that alternative pathways that can induce DDR-mediated cell death, such as CHK1 and/or ATR activation, remain intact.⁴² Activation of ERK1/2, JNK, and p38 determines the sensitivity to therapy in a variety of cancers, including CLL^43,44 and resistance emerging from patients treated with the PI3K inhibitor idelalisib results in elevated MAPK signaling to counteract PI3K pathway inhibition.⁴⁵ Based on our findings, we hypothesize that cells with high levels of ERK signaling would be equally, if not more, vulnerable to DUSP1/6 inhibition, rendering DUSP1/6 inhibition an interesting approach for drug-resistant CLL. Negative regulation of MAPK via DUSP1 (via JNK suppression) also promotes drug resistance in cancer entities,⁴⁶ including osteosarcoma,⁴⁷ lung cancer cells,⁴⁸ and ovarian cancer.⁴⁹ This JNK regulation may contribute to the toxicity induced in CLL by DUSP1/6 inhibition. In clinical patient care, limited responses against the BTK inhibitor ibrutinib are highly relevant; therefore, we investigated whether DUSP1/6 inhibition is also effective against ibrutinib-refractory cases. Based on our findings, these refractory CLL cells are equally vulnerable to DUSP1/6 inhibition in treatment-naïve patients. Therefore, DUSP1/6 inhibition may be equally effective in treating drug-resistant CLL.

Taken together, our findings reveal that in CLL, negative control of MAPK signaling is required to avoid DNA damage response-mediated cell death. We show that high DUSP6 expression is associated with poor clinical outcomes in CLL and may indicate high MAPK signaling, for example, induced by activating mutations of NRAS or BRAF. Furthermore, we demonstrated that inhibiting the activity of DUSP1/6 phosphatases causes DNA damage and cell death in CLL, and that treatment-resistant CLL cases remain sensitive to DUSP1/6 inhibition. As a result, we propose intermittent DUSP1/6 inhibition as a novel approach for CLL therapy, either in combination with kinase inhibition, to potentially boost the efficacy of both solo treatments or to treat kinase inhibitor-resistant disease.

Human subjects. Primary CLL samples were obtained from the peripheral blood of patients treated at the Klinikum rechts der Isar or München Klinik Schwabing, Munich, Germany, or the National Center for Tumor Diseases, Heidelberg, Germany (Suppl. Table 1). Patient sampling was approved by the local ethics committee, and informed consent was obtained from all patients. Healthy donor-derived blood samples were obtained from Bavarian Red Cross (Munich, Germany).

Inhibitor treatment and subsequent analysis. Primary patient-derived CLL cells, MEC-1 cells, B- and T-cell lymphoma, or healthy peripheral blood MACS-isolated B-cells were seeded at 200.000 cells/well in 96-well-plates or 500.000 cells in 24-well plates and treated with BCI (Axon Medchem) alone or in combination with the indicated inhibitors. Viability was analyzed by flow cytometry. All antibodies used are listed in Suppl. Table 2. Please refer to the Supplemental Data for further details.

Mice. We transplanted 2x10e7 splenocytes from aged TCL1-tg mice ²⁸ into C57BL/6 wt immunocompetent mice and waited for the detection of murine CLL cells in the peripheral blood. BCI-215 treatment was initiated daily for a total of 10 doses, and CLL content in the spleen and peritoneal cavity was determined thereafter. Please refer to the Supplemental Data for details.

Phospho-proteome screen. Freshly isolated primary CLL cells were treated with BCI for 3 and 10 min; MEC-1 cells were incubated for 24 h at a cell density of 1x10e6/ml and then treated with BCI for 15 and 45 min before isolating protein. A comprehensive description of the sample processing and subsequent analysis is provided in the Supplemental Data.

All other methods are described in detail in the Supplemental Data.

ACKNOWLEGMENTS:

We would like to thank Tanja Neumeier and Nicole Beck for excellent technical support. This work was funded by the German Cancer Aid (Deutsche Krebshilfe, Max Eder Grant to MB, Project ID:70114720), German Research Foundation (Deutsche Forschungsgemeinschaft, DFG) – Project-ID 360372040 – SFB 1335/P02 awarded to MB, P01 to JR, and Wilhelm Sander Foundation (Project ID 2018.111.1 to MB).

AUTHOR CONTRIBUTIONS:

Veronika Ecker and Lisa Brandmeier: designed and performed the research; Martina Stumpf: designed and performed the research; Piero Giansanti: designed and performed the research; Aida Varela Moreira: designed and performed the research; Lisa Pfeuffer: designed and performed the research; Junyan Lu: analyzed the data; Marcel H. A. M. Fens: designed and performed the research; Bernhard Küster: designed the research; Thomas Engleitner: analyzed the data. Simon Heidegger: contributed CLL samples; Ingo Ringshausen: designed the research; Thorsten Zenz: contributed CLL samples; Clemens-Martin Wendtner: contributed CLL samples; Markus Müschen: designed the research; Jürgen Ruland: designed the research; Maike Buchner: designed the research, performed the research, and wrote the paper.

DATA AVAILABILITY STATEMENT:

The datasets generated during and analyzed during the current study are available from the corresponding author on reasonable request. Phospho-proteome data supporting this publication are deposited in the ProteomeXchange Consortium with the dataset identifier PXD032039.

COMPETING INERESTS:

The authors declare no competing financial interests

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There is NO conflict of interest to disclose.

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Negative feedback regulation of MAPK signaling is an important driver of CLL progression

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Abstract

Figures

Introduction

Results

DUSP1/6 inhibition is toxic specifically for CLL cells

Genetic disruption of DUSP1 or DUSP6 impairs CLL cell expansion

DUSP1/6 inhibition induces BCR/MAPK signaling in CLL cells followed by DNA damage response and apoptosis

Functional relevance of downstream signaling mediated by DUSP1/6 inhibition in CLL

DUSP1/6 inhibition is effective in drug resistant CLL

Discussion

Methods

Declarations

References

Additional Declarations

Supplementary Files

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