Michelle Chen

Rps14 haploinsufficiency causes a block in erythroid differentiation mediated by S100A8 and S100A9.

Impaired erythropoiesis in the deletion 5q (del(5q)) subtype of myelodysplastic syndrome (MDS) has been linked to heterozygous deletion of RPS14, which encodes the ribosomal protein small subunit 14. We generated mice with conditional inactivation of Rps14 and demonstrated an erythroid differentiation defect that is dependent on the tumor suppressor protein p53 (encoded by Trp53 in mice) and is characterized by apoptosis at the transition from polychromatic to orthochromatic erythroblasts. This defect resulted in age-dependent progressive anemia, megakaryocyte dysplasia and loss of hematopoietic stem cell (HSC) quiescence. As assessed by quantitative proteomics, mutant erythroblasts expressed higher levels of proteins involved in innate immune signaling, notably the heterodimeric S100 calcium-binding proteins S100a8 and S100a9. S100a8—whose expression was increased in mutant erythroblasts, monocytes and macrophages—is functionally involved in the erythroid defect caused by the Rps14 deletion, as addition of recombinant S100a8 was sufficient to induce a differentiation defect in wild-type erythroid cells, and genetic inactivation of S100a8 expression rescued the erythroid differentiation defect of Rps14-haploinsufficient HSCs. Our data link Rps14 haploinsufficiency in del(5q) MDS to activation of the innate immune system and induction of S100A8-S100A9 expression, leading to a p53-dependent erythroid differentiation defect.

Core Circadian Clock Genes Regulate Leukemia Stem Cells in AML

Leukemia stem cells (LSCs) have the capacity to self-renew and propagate disease upon serial transplantation in animal models, and elimination of this cell population is required for curative therapies. Here, we describe a series of pooled, in vivo RNAi screens to identify essential transcription factors (TFs) in a murine model of acute myeloid leukemia (AML) with genetically and phenotypically defined LSCs. These screens reveal the heterodimeric, circadian rhythm TFs Clock and Bmal1 as genes required for the growth of AML cells in vitro and in vivo. Disruption of canonical circadian pathway components produces anti-leukemic effects, including impaired proliferation, enhanced myeloid differentiation, and depletion of LSCs. We find that both normal and malignant hematopoietic cells harbor an intact clock with robust circadian oscillations, and genetic knockout models reveal a leukemia-specific dependence on the pathway. Our findings establish a role for the core circadian clock genes in AML.

CrbnI391V is sufficient to confer in vivo sensitivity to thalidomide and its derivatives in mice

Thalidomide and its derivatives, lenalidomide and pomalidomide, are clinically effective treatments for multiple myeloma and myelodysplastic syndrome with del(5q). These molecules lack activity in murine models, limiting investigation of their therapeutic activity or toxicity in vivo. Here, we report the development of a mouse model that is sensitive to thalidomide derivatives because of a single amino acid change in the direct target of thalidomide derivatives, cereblon (Crbn). In human cells, thalidomide and its analogs bind CRBN and recruit protein targets to the CRL4CRBN E3 ubiquitin ligase, resulting in their ubiquitination and subsequent degradation by the proteasome. We show that mice with a single I391V amino acid change in Crbn exhibit thalidomide-induced degradation of drug targets previously identified in human cells, including Ikaros (Ikzf1), Aiolos (Ikzf3), Zfp91, and casein kinase 1a1 (Ck1α), both in vitro and in vivo. We use the Crbn I391V model to demonstrate that the in vivo therapeutic activity of lenalidomide in del(5q) myelodysplastic syndrome can be explained by heterozygous expression of Ck1α in del(5q) cells. We found that lenalidomide acts on hematopoietic stem cells with heterozygous expression of Ck1α and inactivation of Trp53 causes lenalidomide resistance. We further demonstrate that Crbn I391V is sufficient to confer thalidomide-induced fetal loss in mice, capturing a major toxicity of this class of drugs. Further study of the Crbn I391V model will provide valuable insights into the in vivo efficacy and toxicity of this class of drugs.

Abstract 2934: Targeting Bruton's tyrosine kinase with PCI-32765 blocks growth and survival of multiple myeloma and Waldenström macroglobulinemia via potent inhibition of osteoclastogenesis, cytokines/chemokine secretion, and myeloma stem-like cells in the bone marrow microenvironment

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Specific expression of Brutons tyrosine kinase (Btk) in osteoclasts (OC), but not osteoblasts (OB), suggests its role in osteoclastogenesis. Since Btk has not been characterized in multiple myeloma (MM) and Waldenstrom Macroglobulinemia (WM), we investigate effects of PCI-32765, an oral, potent, and selective Btk inhibitor with promising clinical activity in B-cell malignancies, on OC differentiation and function within MM bone marrow (BM) milieu, as well as on MM and WM cancer cells. In CD14+ OC precursor cells, PCI-32765 abrogated RANKL/M-CSF-induced Btk activation and downstream PLCγ2, resulting in decreased number of multinucleated OC (>3 nuclei) by tartrate-resistant acid phosphatase (TRAP) staining and inhibition of TRAP5b (ED50 = 17 nM), a specific mature OC marker. It induced defective bone resorption activity, as evidenced by diminished pit formation on dentine slices. Lack of effect of Dexamethasone on osteoclastic activity was overcome by combination of Dexamethasone with PCI-32765. PCI-32765 potently downregulates cytokine and chemokine secretion from OC cultures, i.e., MIP1α, MIP1α, IL-8, TGFβ1, RANTES, APRIL, SDF-1, and activin A (ED50 = 0.1-0.48 nM). It significantly decreased IL-6, SDF-1, MIP1α, MIP1α, and M-CSF in 2-week cultures of CD138-negative cells from active MM patients, associated with decreased TRAP staining. In MM and WM cells, immunoblotting analysis showed Btk expression in higher percentage of CD138+ myeloma cells from patients (4 out of 5 samples) than MM cell lines (5 out of 9 cell lines), whereas microarray analysis demonstrated increased expression of Btk and its downstream signaling components in WM cells than in CD19+ normal BM cells (p<0.001). Importantly, PCI-32765 blocked SDF-1-induced adhesion and migration of MM cell lines and patient MM cells via blockade of Btk signaling cascade. Quantigene analysis further showed PCI-32765-inhibited MIP1α mRNA in MM cells and many NF-κB-targeted transcripts in OC-lineages. PCI-32765 mitigated MM cell growth and survival triggered by IL-6 and coculture with BM stromal cells (BMSCs) or OCs. It blocked WM cell proliferation and induced apoptosis. Furthermore, myeloma stem-like cells from MM patients expressed Btk and PCI-32765 (10-100 nM) specifically blocked their abilities to form colonies in methylcellulose (n=5). In contrast, no toxicity was observed in Btk-negative BMSCs and OB. Oral administration of PCI-32765 (12 mg/kg) in mice significantly suppressed MM cell growth (p< 0.03) and MM cell-induced osteolysis on implanted human bone chips in a humanized myeloma (SCID-hu) model. Together, these results strongly support clinical trials of targeting Btk by PCI-32765 in the BM microenvironment to improve patient outcome in MM and WM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2934. doi:1538-7445.AM2012-2934