Potent anti-leukemic activity of a specific cyclin-dependent kinase 9 inhibitor in mouse models of chronic lymphocytic leukemia.

Onset of progression even during therapy with novel drugs remains an issue in chronic lymphocytic leukemia (CLL). Thus, there is ongoing demand for novel agents. Approaches targeting cyclin-dependent kinases (CDK) have reached the clinical trial stage. CDK9 mediating RNA transcriptional elongation is the evolving pivotal CLL CDK inhibitor target. However, more CDK9 selective compounds are desirable. Here, we describe the CDK9 inhibitor LDC526 displaying a low nanomolar biochemical activity against CDK9 and an at least 50-fold selectivity against other CDKs. After demonstrating in vitro MEC-1 cell line and primary human CLL cell cytotoxicity we evaluated the LDC526 in vivo effect on human CLL cells transplanted into NOD/scid/γcnull (NSG) mice. LDC526 administration (75 mg/kg) for 5 days resulted in a 77% reduction of human CLL cells in NSG spleens compared to carrier control treatment. Next, we longitudinally studied the LDC526 impact on circulating CLL cells in the TCL1 transgenic mouse model. LDC526 (50 mg/kg) administration for two days led to a 16-fold reduction of blood CLL cell numbers. Remarkably, residual CLL cells exhibited significantly increased intracellular BCL-2 levels. However, the LDC526 cytotoxic effect was not restricted to CLL cells as also declining numbers of normal B and T lymphocytes were observed in LDC526 treated TCL1 mice. Taken together, our in vivo data provide a strong rational for continued LDC526 development in CLL therapy and argue for the combination with BCL-2 inhibitors.

Tumor-derived exosomes modulate PD-L1 expression in monocytes.

In chronic lymphocytic leukemia (CLL), monocytes and macrophages are skewed toward protumorigenic phenotypes, including the release of tumor-supportive cytokines and the expression of immunosuppressive molecules such as programmed cell death 1 ligand 1 (PD-L1). To understand the mechanism driving protumorigenic skewing in CLL, we evaluated the role of tumor cell-derived exosomes in the cross-talk with monocytes. We carried out RNA sequencing and proteome analyses of CLL-derived exosomes and identified noncoding Y RNA hY4 as a highly abundant RNA species that is enriched in exosomes from plasma of CLL patients compared with healthy donor samples. Transfer of CLL-derived exosomes or hY4 alone to monocytes resulted in key CLL-associated phenotypes, including the release of cytokines, such as C-C motif chemokine ligand 2 (CCL2), CCL4, and interleukin-6, and the expression of PD-L1. These responses were abolished in Toll-like receptor 7 (TLR7)-deficient monocytes, suggesting exosomal hY4 as a driver of TLR7 signaling. Pharmacologic inhibition of endosomal TLR signaling resulted in a substantially reduced activation of monocytes in vitro and attenuated CLL development in vivo. Our results indicate that exosome-mediated transfer of noncoding RNAs to monocytes contributes to cancer-related inflammation and concurrent immune escape via PD-L1 expression.

Analysis of Genes Involved in Body Weight Regulation by Targeted Re-Sequencing.

INTRODUCTION: Genes involved in body weight regulation that were previously investigated in genome-wide association studies (GWAS) and in animal models were target-enriched followed by massive parallel next generation sequencing. METHODS: We enriched and re-sequenced continuous genomic regions comprising FTO, MC4R, TMEM18, SDCCAG8, TKNS, MSRA and TBC1D1 in a screening sample of 196 extremely obese children and adolescents with age and sex specific body mass index (BMI) ≥ 99th percentile and 176 lean adults (BMI ≤ 15th percentile). 22 variants were confirmed by Sanger sequencing. Genotyping was performed in up to 705 independent obesity trios (extremely obese child and both parents), 243 extremely obese cases and 261 lean adults. RESULTS AND CONCLUSION: We detected 20 different non-synonymous variants, one frame shift and one nonsense mutation in the 7 continuous genomic regions in study groups of different weight extremes. For SNP Arg695Cys (rs58983546) in TBC1D1 we detected nominal association with obesity (pTDT = 0.03 in 705 trios). Eleven of the variants were rare, thus were only detected heterozygously in up to ten individual(s) of the complete screening sample of 372 individuals. Two of them (in FTO and MSRA) were found in lean individuals, nine in extremely obese. In silico analyses of the 11 variants did not reveal functional implications for the mutations. Concordant with our hypothesis we detected a rare variant that potentially leads to loss of FTO function in a lean individual. For TBC1D1, in contrary to our hypothesis, the loss of function variant (Arg443Stop) was found in an obese individual. Functional in vitro studies are warranted.

Functional abnormalities and changes in gene expression in fibroblasts and macrophages from the bone marrow of patients with acute myeloid leukemia.

In bone marrow malignancies, little is known about the fate of stromal cells after replacement of normal cells by neoplastic hematopoietic ones. In this study, fibroblasts from patients with acute myeloid leukemia or myelodysplastic syndromes exhibited a significantly lower ability to support hematopoiesis originating from co-cultured allogeneic CD34-positive cells than did fibroblasts from healthy marrow. Conversely, macrophages from acute myeloid leukemia marrow significantly enhanced the production of blood cells compared with control macrophages. Aberrant function was associated with consistent changes in the expression of genes involved in hematopoietic stem cell control, such as cytokines and regulators of the Wnt signaling pathway.

Fine Mapping of a GWAS-Derived Obesity Candidate Region on Chromosome 16p11.2.

INTRODUCTION: Large-scale genome-wide association studies (GWASs) have identified 97 chromosomal loci associated with increased body mass index in population-based studies on adults. One of these SNPs, rs7359397, tags a large region (approx. 1MB) with high linkage disequilibrium (r2>0.7), which comprises five genes (SH2B1, APOBR, sulfotransferases: SULT1A1 and SULT1A2, TUFM). We had previously described a rare mutation in SH2B1 solely identified in extremely obese individuals but not in lean controls. METHODS: The coding regions of the genes APOBR, SULT1A1, SULT1A2, and TUFM were screened for mutations (dHPLC, SSCP, Sanger re-sequencing) in 95 extremely obese children and adolescents. Detected non-synonymous variants were genotyped (TaqMan SNP Genotyping, MALDI TOF, PCR-RFLP) in independent large study groups (up to 3,210 extremely obese/overweight cases, 485 lean controls and 615 obesity trios). In silico tools were used for the prediction of potential functional effects of detected variants. RESULTS: Except for TUFM we detected non-synonymous variants in all screened genes. Two polymorphisms rs180743 (APOBR p.Pro428Ala) and rs3833080 (APOBR p.Gly369_Asp370del9) showed nominal association to (extreme) obesity (uncorrected p = 0.003 and p = 0.002, respectively). In silico analyses predicted a functional implication for rs180743 (APOBR p.Pro428Ala). Both APOBR variants are located in the repetitive region with unknown function. CONCLUSION: Variants in APOBR contributed as strongly as variants in SH2B1 to the association with extreme obesity in the chromosomal region chr16p11.2. In silico analyses implied no functional effect of several of the detected variants. Further in vitro or in vivo analyses on the functional implications of the obesity associated variants are warranted.

Progranulin is a novel independent predictor of disease progression and overall survival in chronic lymphocytic leukemia.

Progranulin (Pgrn) is a 88 kDa secreted protein with pleiotropic functions including regulation of cell cycle progression, cell motility, wound repair and tumorigenesis. Using microarray based gene expression profiling we have recently demonstrated that the gene for Pgrn, granulin (GRN), is significantly higher expressed in aggressive CD38(+)ZAP-70(+) as compared to indolent CD38(-)ZAP-70(-) chronic lymphocytic leukemia (CLL) cases. Here, we measured Pgrn plasma concentrations by enzyme-linked immunosorbent assay (ELISA) in the Essen CLL cohort of 131 patients and examined Pgrn for association with established prognostic markers and clinical outcome. We found that high Pgrn plasma levels were strongly associated with adverse risk factors including unmutated IGHV status, expression of CD38 and ZAP-70, poor risk cytogenetics (11q-, 17p-) as detected by flourescence in situ hybridization (FISH) and high Binet stage. Pgrn as well as the aforementioned risk factors were prognostic for time to first treatment and overall survival in this series. Importantly, these results could be confirmed in the independent multicentric CLL1 cohort of untreated Binet stage A patients (n = 163). Here, multivariate analysis of time to first treatment revealed that high risk Pgrn (HR = 2.06, 95%-CI = 1.13-3.76, p = 0.018), unmutated IGHV status (HR = 5.63, 95%-CI = 3.05-10.38, p<0.001), high risk as defined by the study protocol (HR = 2.06, 95%-CI = 1.09-3.89, p = 0.026) but not poor risk cytogenetics were independent prognostic markers. In summary our results suggest that Pgrn is a novel, robust and independent prognostic marker in CLL that can be easily measured by ELISA.