Negative Feedback Regulation of MAPK Signaling Is an Important Driver of CLL Progression

Chronic lymphocytic leukemia (CLL) represents approximately 25% of all new cases of leukemia in the USA. Insight into the pathogenesis of CLL has led to the development of highly effective treatments in the past decade. Nevertheless, CLL patients with activating mutations in the mitogen-activated protein kinase (MAPK) pathway, which enables cell proliferation and differentiation, comprise a distinct subgroup with poor clinical prognosis such as shorter treatment-free survival and higher drug resistance. Dual specificity protein phosphatases (DUSPs) negatively regulate MAPK signaling and are required to limit and terminate the activating signal. Whether DUSP signaling is crucial for the development and progression of MAPK driven CLL is unknown. Thus, in this study we investigated the expression and functional relevance of negative MAPK regulators DUSP1 and DUSP6 in CLL.

Analyzing mRNA data sets of CLL patients, we discovered that DUSP1 and DUSP6 were broadly expressed in CLL. High expression levels of DUSP6, though not DUSP1, were found in cases with activating mutations within the MAPK pathway and correlated with a poor clinical outcome, indicated by shorter time-to-treatment and overall survival.

To investigate the functional relevance of DUSP1- and DUSP6-mediated negative MAPK regulation in CLL, we tested the effects of the dual-specific DUSP1/6 inhibitor BCI on CLL cells. DUSP1/6 inhibition in vitro resulted in a dose-dependent induction of specific cell death of CLL cells, whereas B cells from healthy donors remained unaffected. Treatment of mice with an adoptively transferred murine CLL with an in vivo applicable derivate of BCI also significantly decreased CLL levels in the spleen and peritoneal cavity, compared to a vehicle treated control group. Further, CRISPR/Cas9-mediated knockout of DUSP1 or DUSP6 in the CLL cell line MEC-1 reduced cell proliferation and fitness; however, DUSP1/6 double knockouts lead to syngeneic lethality. Thus, DUSP1 and DUSP6 are crucial for negative MAPK pathway regulation to enable cell survival.

To assess changes in intracellular signaling pathways after DUSP1/6 inhibition, we performed a phospho-proteome analysis of BCI treated patient-derived CLL cells and compared them to vehicle treated cells. As expected, a significant deregulation of B-cell receptor and MAPK signaling was detected after inhibition, with an increase in the activating phosphorylation on ERK1 and ERK2, which are targeted by DUSP1/6, confirming on-target specificity of BCI using an unbiased approach. To gain insight into the mechanism driving cell death, we conducted a BCI time course analysis followed by a phospho-proteome screen and observed that the DNA damage response (DDR) and the apoptosis pathway were among the strongest regulated pathways upon DUSP1/6 inhibition in MEC-1 cells. To validate these findings, we used immunoblotting and detected phosphorylation of DDR key molecules, such as ATF2 and c-JUN, in human CLL samples after DUSP1/6 inhibition. We also used flow cytometry to confirm acute activation of the MAPK pathway after BCI treatment by detecting increased phospho-γH2AX (DNA damage) and phosphatidylserine (apoptosis) levels. Furthermore, we showed that the apoptotic cell death is promoted by CHK1/2 kinases and depends on MAPK activity.

Chemotherapeutic drug resistances are often detected in patients with deletion of p53 or ATM, central regulators of the DDR. Hence, we tested the effectiveness of DUSP1/6 inhibition on patient derived CLLs carrying a loss of function variation of p53 or ATM. We did not detect a significant difference in cytotoxicity, indicating DUSP1/6 inhibition effectively decreases CLL viability, even in cases without functional p53/ATM-mediated DDR. Finally, we generated a MEC-1 cell line resistant to the chemotherapeutic agent idelalisib, as a model for drug resistant CLLs. As the sensitivity to idelalisib decreased, the cells became more sensitive to BCI. When comparing the effects of BCI on CLL cells from treatment naïve patients compared to ibrutinib-refractory patients, we found no differences in sensitivity to DUSP1/6 inhibition.

In conclusion, we show that MAPK deregulation by DUSP1/6 inhibition is highly effective to induce cytotoxic cell death in CLL cells, even without functional p53/ATM-mediated DDR; hence, we suggest DUSP1/6 inhibition is a promising approach to treat drug resistant and refractory CLLs.

Zenz:Abbvie: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Incyte: Consultancy, Honoraria; Janpix: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Roche: Consultancy, Honoraria. Wendtner:Hoffmann-La Roche, Cilag-Janssen, AbbVie: Consultancy, Research Funding.