Background:Chronic inflammation plays a central role in the development of hematological neoplasms. In Myelodysplastic syndromes (MDS) pro‐inflammatory microenvironment is suspected to drive disease development. Several studies have shown increased rates of programmed cell death in marrow cells of patients with early stage MDS. In these patients improvement of cytopenia is a major therapeutic approach. Overcoming resistance to programmed cell death is a central therapeutic aim in patients with high‐risk MDS/sAML. RIPK3 is a crucial player of regulated necrosis (necroptosis). Necroptosis has a strong inflammatory capacity and pro‐inflammatory processes trigger necroptosis.Aims:The aim of this project is to identify pro‐necroptotic proteins in the bone marrow of patients with MDS, CMML and sAML and to correlate the necroptotic capacity with the stage of the disease. By pharmacological modulation of necroptosis we would like to evaluate the effects of necroptotic signaling on inflammation, cell death and cell differentiation.Methods:We therefore analyzed the impact of pro‐necroptotic signaling on bone marrow bulk and the subset of CD34+stem/progenitor cells of patients with MDS and chronic myelomonocytic leukemia (CMML) in vitro. Bone marrow mononuclear cells (BMMNCs) were isolated from BM aspirates of 5 patients with early MDS (MDS‐MLD, MDS‐RS‐SLD and MDS‐RS‐MLD), 5 patients with late MDS (MDS‐EB1 and MDS‐EB2), 5 patients with CMML‐1, 7 patients with CMML‐2 and 4 patients with sAML after a history of MDS and 4 patients with sAML after a history of CMML. Bulk of BMMNCs and purified CD34+stem/progenitor cells were stained intracellular for RIPK3 and analyzed by flow cytometry. The patient samples were compared to 12 age‐matched BM samples obtained from hip replacement surgery from otherwise healthy individuals. Furthermore, immunohistochemistry was performed in an enlarged cohort of patients. Therefore paraffin embedded bone marrow biopsies of individuals with MDS or CMML‐1/2 were analyzed over time and disease progression. Additionally BMMNCs of patients with MDS were treated with specific RIPK1 (Nec1 s) and RIPK3 (GSK 843A) inhibitors for 72 h. Short term viability analysis by flow cytometry were performed and long‐term analysis using growth factor enriched methylcellulose.Results:Expression of RIPK3 was significantly increased in sAML after a history of MDS compared to late MDS and compared to healthy controls. Furthermore patients with sAML after a history of CMML showed a significantly higher expression of RIPK3 than patients with CMML‐1, CMML‐2 and healthy controls. These findings were confirmed by immunohistochemistry in an enlarged cohort of patients. Here paraffin embedded bone marrow biopsies of individuals with MDS or CMML‐1/2 were analyzed over time and disease progression.Additionally BMMNCs of patients with MDS or CMML were treated with specific RIPK1 (Nec1 s) and RIPK3 (GSK 843A) inhibitorsfor 72 h. Short term viability analysis by flow cytometry showed no impact of inhibitor treatment. However after culture in growth factor enriched methylcellulose for 10–14 days colony forming capacity was significantly increased in low‐risk MDS patients after inhibition of pro‐necroptotic signaling. Here increase in colony numbers was not restricted to a specific lineage.Summary/Conclusion:We conclude that RIPK3 plays a critical stage‐dependent role in patients with MDS and CMML. Disease progression to sAML is associated with an increased RIPK3‐Expression. We further hypothesize that pro‐inflammatory processes like necroptosis have an impact on aberrant myeloid homeostasis.

Deregulated apoptosis is an identifying feature of myelodysplastic syndromes (MDS). Whereas apoptosis is increased in the bone marrow (BM) of low-risk MDS patients, progression to high-risk MDS correlates with an acquired resistance to apoptosis and an aberrant expression of BCL-2 proteins. To overcome the acquired apoptotic resistance in high-risk MDS, we investigated the induction of apoptosis by inhibition of pro-survival BCL-2 proteins using the BCL-2/-X L /-W inhibitor ABT-737 or the BCL-2-selective inhibitor ABT-199. We characterized a cohort of 124 primary human BM samples from MDS/secondary acute myeloid leukemia (sAML) patients and 57 healthy, age-matched controls. Inhibition of anti-apoptotic BCL-2 proteins was specifically toxic for BM cells from high-risk MDS and sAML patients, whereas low-risk MDS or healthy controls remained unaffected. Notably, ABT-737 or ABT-199 treatment was capable of targeting the MDS stem/progenitor compartment in high-risk MDS/sAML samples as shown by the reduction in CD34 + cells and the decreased colony-forming capacity. Elevated expression of MCL-1 conveyed resistance against both compounds. Protection by stromal cells only partially inhibited induction of apoptosis. Collectively, our data show that the apoptotic resistance observed in high-risk MDS/sAML cells can be overcome by the ABT-737 or ABT-199 treatment and implies that BH3 mimetics might delay disease progression in higher-risk MDS or sAML patients.

Penile squamous cell carcinoma (PSCC) is a rare tumor associated with high-risk human papillomavirus (HR-HPV) infection in 30% to 60% of cases. Altered expression of miRNAs has been reported in HPV-related cervical and head and neck cancers, but such data have not been available for PSCC. We analyzed a series of 59 PSCCs and 8 condylomata for presence of HPV infection, for p16INK4a, Ki-67, and p53 immunohistochemical expression, and for expression of a panel of cellular miRNAs (let-7c, miR-23b, miR-34a, miR-145, miR-146a, miR-196a, and miR-218) involved in HPV-related cancer. HR-HPV DNA (HPV16 in most cases) was detected in 17/59 (29%) PSCCs; all penile condylomata (8/8) were positive for low-risk HPV6 or HPV11. HR-HPV+ PSCCs overexpressed p16INK4a in 88% cases and p53 in 35% of cases, whereas HR-HPV− PSCCs were positive for p16INK4a and p53 immunostaining in 9% and 44% of cases, respectively. Among the miRNAs investigated, expression of miR-218 was lower in PSCCs with HR-HPV infection and in p53− cancers. Hypermethylation of the promoter of the SLIT2 gene, which contains miR-218-1 in its intronic region, was frequently observed in PSCCs, mainly in those with low miR-218 expression. Epigenetic silencing of miR-218 is a common feature in HR-HPV+ PSCCs and in HR-HPV− PSCCs without immunohistochemical detection of p53.

Chronic lymphocytic leukemia (CLL) has a high prevalence in western countries and remains incurable to date. Here, we provide evidence that the multikinase inhibitor sorafenib induces apoptosis in primary CLL cells. This strong pro-apoptotic effect is not restricted to any subgroup of patients, based on Binet stage and the expression of ZAP70 or CD38. Mechanistically, sorafenib-induced cell death is preceded by a rapid downregulation of Mcl-1 through the inhibition of protein translation. Subsequently, the cell intrinsic apoptotic pathway is activated, indicated by destabilization of the mitochondrial membrane potential and activation of caspase-3 and -9. In contrast to sorafenib, the monoclonal vascular epidermal growth factor (VEGF)-antibody bevacizumab failed to induce apoptosis in CLL cells, suggesting that sorafenib induces cell death irrespectively of VEGF signalling. Notably, although sorafenib inhibits phosphorylation of the Scr-kinase Lck, knock-down of Lck did not induce apoptosis in CLL cells. Of note, the pro-apoptotic effect of sorafenib is not restricted to cell-cycle arrested cells, but is also maintained in proliferating CLL cells. In addition, we provide evidence that sorafenib can overcome drug resistance in CLL cells protected by microenvironmental signals from stromal cells. Conclusively, sorafenib is highly active in CLL and may compose a new therapeutic option for patients who relapse after immunochemotherapy.