Mindaugas Andrulis

A complex secretory program orchestrated by the inflammasome controls paracrine senescence

Oncogene-induced senescence (OIS) is crucial for tumour suppression. Senescent cells implement a complex pro-inflammatory response termed the senescence-associated secretory phenotype (SASP). The SASP reinforces senescence, activates immune surveillance and paradoxically also has pro-tumorigenic properties. Here, we present evidence that the SASP can also induce paracrine senescence in normal cells both in culture and in human and mouse models of OIS in vivo. Coupling quantitative proteomics with small-molecule screens, we identified multiple SASP components mediating paracrine senescence, including TGF-β family ligands, VEGF, CCL2 and CCL20. Amongst them, TGF-β ligands play a major role by regulating p15INK4b and p21CIP1. Expression of the SASP is controlled by inflammasome-mediated IL-1 signalling. The inflammasome and IL-1 signalling are activated in senescent cells and IL-1α expression can reproduce SASP activation, resulting in senescence. Our results demonstrate that the SASP can cause paracrine senescence and impact on tumour suppression and senescence in vivo.

BRAF Inhibition in Refractory Hairy-Cell Leukemia

The authors report a dramatic response to vemurafenib in a patient with hairy-cell leukemia refractory to nucleosides and rituximab.

Targeting the BRAF V600E Mutation in Multiple Myeloma

In multiple myeloma, there has been little progress in the specific therapeutic targeting of oncogenic mutations. Whole-genome sequencing data have recently revealed that a subset of patients carry an activating mutation (V600E) in the BRAF kinase. To uncover the clinical relevance of this mutation in multiple myeloma, we correlated the mutation status in primary tumor samples from 379 patients with myeloma with disease outcome. We found a significantly higher incidence of extramedullary disease and a shorter overall survival in mutation carriers when compared with controls. Most importantly, we report on a patient with confirmed BRAF V600E mutation and relapsed myeloma with extensive extramedullary disease, refractory to all approved therapeutic options, who has rapidly and durably responded to low doses of the mutation-specific BRAF inhibitor vermurafenib. Collectively, we provide evidence for the development of the BRAF V600E mutation in the context of clonal evolution and describe the prognostic and therapeutic relevance of this targetable mutation.

Application of a BRAF V600E mutation-specific antibody for the diagnosis of hairy cell leukemia.

In recent times BRAF V600E mutations have emerged as a genetic hallmark of hairy cell leukemia (HCL). This specific point mutation is present in virtually all cases of HCL but is exceedingly rare in other peripheral B-cell neoplasms. In this study we investigated the application of a BRAF V600E mutation-specific antibody (clone VE1) to differentiate HCL from HCL mimics, such as HCL variant and splenic marginal zone lymphoma. A total of 52 routinely processed formalin-fixed paraffin-embedded tissue specimens were investigated (bone marrow, n=46; spleen, n=6) for expression of V600E-mutated BRAF protein. All 32 cases of HCL were scored positive, and all non-HCL cases were scored negative. In 28 of 30 HCL cases the presence of a BRAF V600E mutation could be confirmed by direct sequencing, whereas no BRAF mutations were detected among 20 HCL mimics. We further screened 228 mature B-cell neoplasms with VE1 and detected 1 positive case of chronic lymphocytic leukemia. Sequencing confirmed the presence of a BRAF V600E mutation. In conclusion, we demonstrate that VE1 immunohistochemistry can be used to reliably differentiate HCL from HCL-mimicking entities. This on-slide technique might be particularly helpful in interpreting challenging biopsies with low tumor content.

The PI3K/Akt signaling pathway regulates the expression of Hsp70, which critically contributes to Hsp90-chaperone function and tumor cell survival in multiple myeloma

Despite therapeutic advances multiple myeloma remains largely incurable, and novel therapeutic concepts are needed. The Hsp90-chaperone is a reasonable therapeutic target, because it maintains oncogenic signaling of multiple deregulated pathways. However, in contrast to promising preclinical results, only limited clinical efficacy has been achieved through pharmacological Hsp90 inhibition. Because Hsp70 has been described to interact functionally with the Hsp90-complex, we analyzed the suitability of Hsp72 and Hsp73 as potential additional target sites. Expression of Hsp72 and Hsp73 in myeloma cells was analyzed by immunohistochemical staining and western blotting. Short interfering RNA-mediated knockdown or pharmacological inhibition of Hsp72 and Hsp73 was performed to evaluate the role of these proteins in myeloma cell survival and for Hsp90-chaperone function. Furthermore, the role of PI3K-dependent signaling in constitutive and inducible Hsp70 expression was investigated using short interfering RNA-mediated and pharmacological PI3K inhibition. Hsp72 and Hsp73 were frequently overexpressed in multiple myeloma. Knockdown of Hsp72 and/or Hsp73 or treatment with VER-155008 induced apoptosis of myeloma cells. Hsp72/Hsp73 inhibition decreased protein levels of Hsp90-chaperone clients affecting multiple oncogenic signaling pathways, and acted synergistically with the Hsp90 inhibitor NVP-AUY922 in the induction of death of myeloma cells. Inhibition of the PI3K/Akt/GSK3β pathway with short interfering RNA or PI103 decreased expression of the heat shock transcription factor 1 and down-regulated constitutive and inducible Hsp70 expression. Treatment of myeloma cells with a combination of NVP-AUY922 and PI103 resulted in additive to synergistic cytotoxicity. In conclusion, Hsp72 and Hsp73 sustain Hsp90-chaperone function and critically contribute to the survival of myeloma cells. Translation of Hsp70 inhibition into the clinic is therefore highly desirable. Treatment with PI3K inhibitors might represent an alternative therapeutic strategy to target Hsp70.

Diffusion-weighted imaging for non-invasive and quantitative monitoring of bone marrow infiltration in patients with monoclonal plasma cell disease: A comparative study with histology

Bone marrow plasma cell infiltration is a crucial parameter of disease activity in monoclonal plasma cell disorders. Until now, the only way to quantify such infiltration was bone marrow biopsy or aspiration. Diffusion‐weighted imaging (DWI) is a magnetic resonance imaging‐technique that may mirror tissue cellularity by measuring random movements of water molecules. To investigate if DWI is capable of assessing bone marrow cellularity in monoclonal plasma cell disease, we investigated 56 patients with multiple myeloma or monoclonal gammopathy of undetermined significance, and 30 healthy controls using DWI of the pelvis and/or the lumbar spine. In 25 of 30 patients who underwent biopsy, bone marrow trephine and DWI could be compared. Of the patients with symptomatic disease 15 could be evaluated after systemic treatment. There was a positive correlation between the DWI‐parameter apparent diffusion coefficient (ADC) and bone marrow cellularity as well as micro‐vessel density (P < 0·001 respectively). ADC was significantly different between patients and controls (P < 0·01) and before and after systemic therapy (P < 0·001). In conclusion, DWI enabled bone marrow infiltration to be monitored in a non‐invasive, quantitative way, suggesting that after further investigations on larger patient groups this might become an useful tool in the clinical work‐up to assess tumour burden.

Combined functional and molecular analysis of tumor cell signaling defines 2 distinct myeloma subgroups: Akt-dependent and Akt-independent multiple myeloma

Although the phosphatidylinositide 3-kinase (PI3K)/Akt pathway has been reported to contribute to the malignant growth of multiple myeloma (MM), the true relevance of Akt kinases for this disease is still unclear. In particular, functional analyses in primary tumor cells and genetic target validation experiments are missing. Here, we used combined functional and molecular analyses to determine the importance of Akt activity in a large panel of primary MM samples and in MM cell lines. Akt down-regulation with isoform-specific siRNA constructs or with an Akt1/2-specific pharmacologic inhibitor strongly induced apoptosis in approximately half of the primary MM samples analyzed. Sensitivity to Akt inhibition strongly correlated with the activation status of Akt as determined by immunohistochemistry, phospho-Akt-specific flow cytometry, and Western analysis. Additional blockade of the MAPK and the IL-6R/STAT3 pathways was often not sufficient to decrease the viability of MM cells resilient to Akt inhibition. Taken together, these experiments led to the identification of 2 myeloma subgroups: Akt-dependent and Akt-independent MM.

Blastic Plasmacytoid Dendritic Cell Neoplasia (BPDC) in Elderly Patients: Results of a Treatment Algorithm Employing Allogeneic Stem Cell Transplantation with Moderately Reduced Conditioning Intensity

Blastic plasmacytoid dendritic cell neoplasm (BPDC), formerly known as blastic NK cell lymphoma, is a rare hematopoietic malignancy preferentially involving skin, bone marrow, and lymph nodes. The overall prognosis of BPDC is dismal, with a median overall survival (OS) of only 12 to 14 months despite aggressive chemotherapy. Anecdotal reports suggest that younger patients might benefit from myeloablative therapy with autologous or allogeneic stem cell transplantation (alloSCT). However, with a median age at diagnosis beyond 60 years, BPDC primarily affects elderly patients. Here, we present for the first time evidence that also in elderly patients, alloSCT for BPDC is feasible and may result in sustained remission if conditioning with moderately reduced intensity is used. Between 2006 and 2009, 6 patients were treated at our institution who fulfilled the diagnostic criteria for BPDC. Median age was 67 (range: 55-80) years. All responded to acute leukemia-type induction therapy. Whereas 2 patients who were ineligible for alloSCT rapidly died of disease recurrence, 4 patients underwent alloSCT from unrelated donors as part of first-line (n = 1) or salvage treatment (n = 3). Two patients allografted in remission live disease free 57 and 16 months post-alloSCT, whereas 2 patients transplanted with active disease achieved complete remission but relapsed 6 and 18 months after transplantation, respectively. In conclusion, reduced-intensity conditioning (RIC) alloSCT from unrelated donors is feasible and seems to be effective in elderly patients with BPDC, suggesting that alloSCT should be pursued aggressively in patients with this otherwise fatal disease up to 70 years of age.

Endothelial Vulnerability and Endothelial Damage Are Associated with Risk of Graft-versus-Host Disease and Response to Steroid Treatment

There is accumulating evidence indicating that endothelial factors are involved in the pathogenesis of GVHD. We have recently shown that steroid-refractory, but not sensitive, GVHD is characterized by higher pretransplantation serum levels of angiopoetin-2 (ANG2), a hormone mediating endothelial vulnerability. To evaluate whether endothelial vulnerability is a risk factor for GVHD per se or becomes important only when noticeable GVHD is established, we measured ANG2 along with additional serum markers of endothelial stress, including soluble thrombomodulin (sTM), IL-8 (CXCL8), and hepatocyte growth factor (HGF), in patients with no, low-grade, or severe GVHD. Patients with refractory GVHD exhibited elevated serum levels of ANG2, sTM, HGF, and IL-8 posttransplantation compared with patients with sensitive GVHD and patients without GVHD. Pretransplantation ANG2 was the only growth factor correlated with the risk of refractoriness and mortality, and then only within the subset of patients who developed grade III-IV GVHD. In contrast, ANG2 was not predictive of GVHD or nonrelapse mortality (NRM) in patients with no GVHD or low-grade GVHD. These findings provide evidence that endothelial function plays an important role in the pathogenesis of steroid refractoriness in ongoing GVHD; however, endothelial vulnerability does not predict incidence of GVHD.

SUMOylation Interferes with CCAAT/Enhancer-Binding Protein β-Mediated c-myc Repression, but Not IL-4 Activation in T Cells

The transcription factor C/EBPβ transactivates the IL-4 gene in murine T lymphocytes and facilitates Th2 cell responses. In this study, we demonstrate that C/EBPβ also acts as a repressor of T cell proliferation. By binding to the c-myc promoter(s), C/EBPβ represses c-Myc expression and, therefore, arrests T cells in the G1 phase of the cell cycle. For C/EBPβ-mediated repression, the integrity of its N-terminal transactivation domain is essential whereas the central regulatory domain is dispensable. This central regulatory domain is sumoylated in vivo which leads to an alteration of the activity of C/EBPβ. Whereas sumoylation does not affect the C/EBPβ-mediated activation of the IL-4 gene, it relieves its repressive effect on c-Myc expression and T cell proliferation. Similar to several other transcription factors, sumoylation redistributes nuclear C/EBPβ and targets it to pericentric heterochromatin. These results suggest an important role of sumoylation in adjusting the finely tuned balance between proliferation and differentiation in peripheral T cells which is controlled by C/EBPβ.