Serine/Threonine Kinase STK4 Is a Novel Target in Myeloma

Multiple myeloma (MM) is a disease characterized by clonal proliferation of plasma cells with a complex genomic phenotype. We recently demonstrated that MM cells have signs of ongoing DNA damage and activation of DNA damage response, via ATM phosphorylation and nuclear localization of ABL1 (Cottini et al, Nature Medicine, 2014). In normal cells, the activation of ATM/ABL1 pathway eventually results in the death of cells with terminal unrepaired DNA damage. However, MM cells inactivate YAP1, a downstream target of ABL1, by homozygous deletions in 10 percent of patients or by protein/mRNA downregulation. Re-expression of YAP1 induces growth inhibition and cell death in YAP1 deleted-cell lines (KMS-18 and KMS-20), as well as in cell lines with low YAP1 expression (MM.1S), according to an ABL1-dependent mechanism. In the presence of DNA damage, YAP1 stabilizes and binds to TP73, facilitating the activation of TP73-target genes, such as BAX, PUMA, and CDKN1A (p21). TP73 levels are generally low in MM cells, indicating that YAP1 is the main regulator of TP73; low YAP1 therefore blocks DNA damage-mediated apoptosis. We identified that YAP1 expression is tightly regulated by the activation of the Hippo pathway serine/threonine kinase STK4. Upon STK4 silencing, YAP1 expression was increased, causing MM cell death both in in vitro and in vivo settings. A kinase screening was performed and we are currently optimizing different compounds with anti-STK4 activity. One of them (STK4i) showed promising growth inhibitory effects. To characterize the efficacy of STK4i, a panel of MM cell lines was incubated with increasing doses of the compound ranging from 0.1-10 μM; growth inhibitory effects were measured by MTT assay, while Annexin V-PI staining detected apoptosis. KSM-20 MM cells, which bear a homozygous deletion for YAP1 genomic locus, were used as negative control. After 48 hour treatment with 1μM dose of STK4i, 62 percent, 66 percent and 15 percent of MM.1S, H929 and U266 cells were positive for Annexin V and PI markers, while minimal effects were observed in KMS-20 cells. To test the activation of YAP1 axis in MM, cells were treated with STK4i for 48hrs, and western blot analysis followed, which confirmed YAP1 upregulation in MM.1S and H929 cells. Further studies are ongoing to evaluate additional functions of STK4 in MM growth and to better characterize the activity of STK4i in the bone marrow microenvironment. Importantly, STK4 silencing also upregulates TP73, in a YAP1-dependent manner. To investigate synergic effects of TP73 and TP53, we used STK4 silencing or STK4i in combination with Nutlin-3a, a MDM2 inhibitor that activates TP53, in both wild-type and mutant TP53 cells. A cooperative response in terms of toxicity by combined TP73 and TP53 was observed in TP53 wild type MM cell lines (MM.1S and H929) as well as in OPM-2 cells with mutant-TP53. However, the response in mutant-TP53 patient cells may be variable, depending on residual TP53 function or dominant negative activity of mutant-TP53 on TP73. To conclude, STK4 is a new and important target in MM pathogenesis and STK4 inhibitors may represent novel therapeutic options in a broad subset of patients with MM.