Citations

AbstractTo identify rational therapeutic combinations with cytarabine (Ara-C), we developed a high-throughput, small-interference RNA (siRNA) platform for myeloid leukemia cells. Of 572 kinases individually silenced in combination with Ara-C, silencing of 10 (1.7%) and 8 (1.4%) kinases strongly increased Ara-C activity in TF-1 and THP-1 cells, respectively. The strongest molecular concepts emerged around kinases involved in cell-cycle checkpoints and DNA-damage repair. In confirmatory siRNA assays, inhibition of WEE1 resulted in more potent and universal sensitization across myeloid cell lines than siRNA inhibition of PKMYT1, CHEK1, or ATR. Treatment of 8 acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and chronic myeloid leukemia (CML) cell lines with commercial and the first-in-class clinical WEE1 kinase inhibitor MK1775 confirmed sensitization to Ara-C up to 97-fold. Ex vivo, adding MK1775 substantially reduced viability in 13 of 14 AML, CML, and myelodysplastic syndrome patient samples compared with Ara-C alone. Maximum sensitization occurred at lower to moderate concentrations of both drugs. Induction of apoptosis was increased using a combination of Ara-C and MK1775 compared with using either drug alone. WEE1 is expressed in primary AML, ALL, and CML specimens. Data from this first siRNA-kinome sensitizer screen suggests that inhibiting WEE1 in combination with Ara-C is a rational combination for the treatment of myeloid and lymphoid leukemias.

While no specific genetic markers are required in the diagnosis of multiple myeloma (MM), multiple genetic abnormalities and gene signatures are used in disease prognostication and risk stratification. This is particularly important for the adequate identification of the high-risk MM group, which does not benefit from any of the current therapies, and novel approaches need to be proposed. Fluorescence in situ hybridization (FISH) has been employed for establishing risk-based stratification and still remains the most used genetic technique in the clinical routine. The incorporation of gene expression profiling (GEP) in the study of MM has shown to be a very powerful test in the patient stratification, but its incorporation in clinical routine depends on some technical and logistic resolutions. Thus, FISH still remains the gold standard test for detecting genomic abnormalities and outcome discrimination in MM.

Abstract The precise molecular mechanism of action and targets through which thalidomide and related immunomodulatory drugs (IMiDs) exert their antitumor effects remains unclear. We investigated the role of cereblon (CRBN), a primary teratogenic target of thalidomide, in the antimyeloma activity of IMiDs. CRBN depletion is initially cytotoxic to human myeloma cells, but surviving cells with stable CRBN depletion become highly resistant to both lenalidomide and pomalidomide, but not to the unrelated drugs bortezomib, dexamethasone, and melphalan. Acquired deletion of CRBN was found to be the primary genetic event differentiating isogenic MM1.S cell lines cultured to be sensitive or resistant to lenalidomide and pomalidomide. Gene expression changes induced by lenalidomide were dramatically suppressed in the presence of CRBN depletion, further demonstrating that CRBN is required for lenalidomide activity. Downstream targets of CRBN include interferon regulatory factor 4 (IRF4) previously reported to also be a target of lenalidomide. Patients exposed to, and putatively resistant to, lenalidomide had lower CRBN levels in paired samples before and after therapy. In summary, CRBN is an essential requirement for IMiD activity and a possible biomarker for the clinical assessment of antimyeloma efficacy.

Abstract Purpose: Only a limited number of genetic studies have been conducted in primary central nervous system lymphomas (PCNSL), partly due to the rarity of the tumors and the very limited amount of available tissue. In this report, we present the first molecular characterization of copy number abnormalities (CNA) of newly diagnosed PCNSL by array-based comparative genomic hybridization (aCGH) in formalin-fixed paraffin-embedded (FFPE) specimens and compare the results with matched, frozen tumor specimens. Experimental Design: We conducted aCGH in FFPE tissues from PCNSL. Results were compared with matched, paired, frozen tumors. Results: Our analysis confirmed the good to fair quality and reliability of the data generated from limited amounts of tumoral FFPE tissue. Overall, all PCNSL cases were characterized by highly complex karyotypes, with a median of 23 CNAs per patient (range, 17–47). Overall, 20 chromosomal regions were recurrently found in more than 40% of cases. Deletions of 6p21, 6q, and 9p21.3 and gain of 12q12-q24.33 were the commonest CNAs. Other minimal affected regions were defined, and novel recurrent CNAs affecting single genes were identified in 3q26.32 (TBL1XR1) and 8q12.1 (TOX). Conclusions: The results obtained are encouraging. Larger archival tissue collections can now be analyzed to complement the still fragmented knowledge we have of the genetic basis of the PCNSL. Clin Cancer Res; 17(13); 4245–53. ©2011 AACR.

Abstract Waldenström macroglobulinemia (WM) is a rare, lymphoplasmacytic lymphoma characterized by hypersecretion of immunoglobulin M (IgM) protein and tumor infiltration into the bone marrow and lymphatic tissue. Our understanding of the mechanisms driving the development and progression of WM is currently by the shortage of representative cell models available for study. We describe here the establishment of a new WM cell line, MWCL-1. Comprehensive genetic analyses have unequivocally confirmed a clonal relationship between this novel cell line and the founding tumor. MWCL-1 cells exhibit an immunophenotype consistent with a diverse, tumor clone composed of both small B lymphocytes and larger lymphoplasmacytic cells and plasma cells: CD3−, CD19+, CD20+, CD27+, CD38+, CD49D+, CD138+, cIgM+, and κ+. Cytogenetic studies identified a monoallelic deletion of 17p13 (TP53) in both the cell line and the primary tumor. Direct DNA resequencing of the remaining copy of TP53 revealed a missense mutation at exon 5 (V143A, GTG>GCG). In accordance with primary WM tumors, MWCL-1 cells retain the ability to secrete high amounts of IgM protein in the absence of an external stimulus. The genetic, immunophenotypic, and biologic data presented here confirm the validity of the MWCL-1 cell line as a representative model of WM.

Abstract Introduction. PCNSL is an aggressive primary brain tumor characterized by a perivascular accumulation of malignant cells that have lymphoid characteristics. As there is significantly less information regarding the molecular pathogenesis of PCNSL in comparison with systemic lymphomas the aim of this study was to perform a comprehensive high-resolution genomic analysis in a cohort of PCNSL cases to better characterize the disease at chromosomal and gene level. Material and Methods. Specimens from 7 EBV- and HIV-negative and presumed immunocompetent PCNSL patients with sufficient frozen tissue for DNA extraction were studied. Copy-number abnormalities were analyzed by array CGH using SurePrint G3 (1 million probes) microarray (Agilent). B-cell differentiation status was characterized by immunostains for CD10, MUM-1, and CD138. Results. PCNSL cases were characterized by highly complex genomes with a median of 23 copy-number abnormalities per patient (range 16-49). Twenty-four chromosomal regions were affected in recurrent deletions and 19 in copy-number gains. Gain of 12q12-q24.33 and losses of 9p21.3 and 6q were the most common abnormalities, found in five of the seven patients. There was not a deleted region on 6q unique to all cases, with 6q14.1-q14.3, 6q16.3-q22.2 and 6q25.3-q26 losses being found in 4 out of 5 cases with 6q deletions. Two regions of interest were biallelically deleted. CDKN2A was the sole gene included in all cases with 9p21.3, being biallelically deleted in two out of 5 cases. TOX and CD58, genes associated with the development and activation of T cells, were also biallelically affected. Focal monoallelic deletions affecting negative regulators of the NFkB signaling pathway (MAP4K1, TANK, TAX1BP1, TRIB3), cell cycle (RB1) and immune-cell regulation (SIRPB1, CBLB, NFATC2) were also identified. Overall, a total of 22 genes were included in homozygous deletions in at least one case. Several chromosomal breakpoints were identified inside genes that encode transcription factors previously identified as being part of fusion protein in other hematological malignancies (FOXP1, ETV6 and NCOA2). Conclusion. This pilot study showed evidence for a highly complex genome, mainly characterized by abnormalities affecting a plethora of genes involved in lymphocyte development, activation and NF-kB signaling pathways regulation. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 331.

Abstract Microarray technologies are powerful assays, which can identify copy-number abnormalities (CNA) underlying the pathogenesis of cancer. Several commercial arrays are available but it is unclear which is the best platform. We did a side-by-side comparison between two leading arrays; the Affymetrix SNP6 and Agilent 1M, to compare data compression, noise, gene and exon coverage, and CNA detection. Five samples were run on both platforms and analyzed as recommended by the manufacturer. We first performed an in silico comparison to define the number of probes per serial 5k interval, number of probes per gene and exon, and the coefficient of variation of different smoothing windows. Comparing the CV of the two arrays showed a similar level of variation between a 3 and 5 probe averaging on the 1M and SNP6, respectively. Using these smoothing levels the 1M interrogates 81.2% of the genes in the genome compared to 69.3% on the SNP6. At the exon level the 1M has at least one probe in 64.8% compared to 41.2% on the SNP6. We observed a similar level of data compression (∼10%) on the two arrays but the SNP6 had nearly twice the noise (SD 0.38 versus 0.21). Finally, there was a marked increment in the number of CNA identified by 1M (mean of 78.5) than SNP6.0 (59.5). In this comparison the Agilent 1M out performed the Affymetrix SNP6. Even with half the number of probes, the 1M was superior in CNA detection, gene/exon coverage and showed less signal variation. A comparison with previous platforms showed that these two arrays did not identify new gene amplifications but did detect new deletions, however, neither platform detected several known deletions detected by other methods. Therefore, we designed a custom, exon-centered, 2×400K array (Agilent) with the goal of identifying additional deletions. The design outline was: a) all the probes from the Agilent CGH 44K array; b) 3 probes per exon across the human exome; c) high resolution tiling of 1450 cancer implicated genes; and d) tiling the 4kb flanking all MIRs. To improve performance, we only included probes with performance scores higher than 0.8 and 0.85 in queries b) and c), respectively. Lastly, 15000 probes from the 1M array that map to exons missed by our design parameters were also included. Our custom array has half the probes (415K) of the 1M (960K) and 4.5x less than the SNP6 (1.85M) but shows the best gene and exon coverage. When the minimum number of probes needed for calculating a CNA is considered, 86% of genes are interrogated compared to 81.2% (1M) and 69.3% (SNP6). The difference is even more marked at the exon level with 84% covered by at least one probe and at least 63.2% interrogated by two probes compared to 5.3% (1M) and 18.0% (SNP6). The promising in silico comparison of this exon-centered array, its more stringent design parameters in combination with significantly reducing the cost per run should make this a very promising array design for CNA analysis of tumor samples in the future Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4957.

Abstract The molecular target(s) cooperating with proteasome inhibition in multiple myeloma (MM) remain unknown. We therefore measured proliferation in MM cells transfected with 13 984 small interfering RNAs in the absence or presence of increasing concentrations of bortezomib. We identified 37 genes, which when silenced, are not directly cytotoxic but do synergistically potentiate the growth inhibitory effects of bortezomib. To focus on bortezomib sensitizers, genes that also sensitized MM to melphalan were excluded. When suppressed, the strongest bortezomib sensitizers were the proteasome subunits PSMA5, PSMB2, PSMB3, and PSMB7 providing internal validation, but others included BAZ1B, CDK5, CDC42SE2, MDM4, NME7, RAB8B, TFE3, TNFAIP3, TNK1, TOP1, VAMP2, and YY1. The strongest hit CDK5 also featured prominently in pathway analysis of primary screen data. Cyclin-dependent kinase 5 (CDK5) is expressed at high levels in MM and neural tissues with relatively low expression in other organs. Viral shRNA knockdown of CDK5 consistently sensitized 5 genetically variable MM cell lines to proteasome inhibitors (bortezomib and carfilzomib). Small-molecule CDK5 inhibitors were demonstrated to synergize with bortezomib to induce cytotoxicity of primary myeloma cells and myeloma cell lines. CDK5 regulation of proteasome subunit PSMB5 was identified as a probable route to sensitization.

Abstract Multiple myeloma (MM) is a cancer of the plasma cells in the bone marrow. Although rare, MM is a deadly form of cancer with a 5 year survival rate of only 37%. African Americans (AA) are twice as likely to be diagnosed with and die from MM as compared to European Americans (EA). Socioeconomic factors may contribute to this disparity, however, biological factors may also be an influence. We have identified somatic copy number alterations in MM as a part of the Multiple Myeloma Genomics Initiative by profiling 250 MM tumors using array-based comparative genomic hybridization (aCGH). Using this cohort, we identified 16 African American and 180 European American patients using a MMRC database that indicated the racial demographics of each patient. To determine if somatic alterations may play a role in the disparity in incidence and/or severity of MM in AA, we compared the frequency of specific chromosomal aberrations in genes and genomic regions found to be involved in progression and poor outcome. The number of events of occurring was calculated and a univariate analysis was conducted using the Fisher's exact test. P-values were used to determine statistical significance. Results revealed significant differences for chromosome 1 p alterations. Specifically, homozygous deletion at the p18 locus at 1 p occurred in 12.5% (2/16) of tumors from AA patients and 2.8% (5/180) of EA patient tumors (p = 0.045). Further stratification of tumors by hyperdiploid or non-hyperdiploid status revealed significant differences in p18 homozygous deletion. Homozygous deletion of p18 was found in 25% (2/8) of AA patients and only 1.2% (1/83) of EA patients with non-hyperdiploid tumors (p=3.2 × 10-4). A nominally significant difference among non-hyperdiploid patients was also present for overall 1 p deletion, which was observed in 2/8 (25%) of AA non-hyperdiploid tumors and 33/83 (40%) of EA with non-hyperdiploid tumors (p=0.05). No differences were observed for 1 p deletion or p18 homozygous deletion among hyperdiploid tumors between AA and EA patients. We then combined our AA dataset with an independent dataset of 8 AA MM patients generated at the Mayo Clinic, Scottsdale AZ and repeated the analysis. Interestingly, very little or no aberrations were found in the dataset from the Mayo Clinic, however, differences of p18 homozygous deletion and 16q deletion among AA and EA remained statistically significant. To determine the correlation between hemizygous and biallelic loss of p18 and poor prognosis we used the 70 gene signature (Shaughnessy, et al., 2007) as a surrogate to identify patients with poor prognosis within the Multiple Myeloma Genomics Initiative dataset. Genomic identification of significant targets in cancer (GISTIC) was used (Beroukhim et al., 2007) to identify regions of aberrant copy number that were over-represented among samples. A relationship between poor prognosis and biallelic loss was found to be statistically significant (p = 0002) while samples with hemizygous loss showed no relationship (p = 0.57). Although the size of the current AA cohort is small, these results suggest that homozygous deletions at the p18 locus may be more common among African American MM patients with non-hyperdiploid tumors which may help to explain the disparity in AA patients with MM. Array CGH is currently being conducted on an additional 40 tumors from AA MM patients for a more robust analysis. Citation Information: Cancer Epidemiol Biomarkers Prev 2010;19(10 Suppl):B64.

AbstractA paucity of validated kinase targets in human multiple myeloma has delayed clinical deployment of kinase inhibitors in treatment strategies. We therefore conducted a kinome-wide small interfering RNA (siRNA) lethality study in myeloma tumor lines bearing common t(4;14), t(14;16), and t(11;14) translocations to identify critically vulnerable kinases in myeloma tumor cells without regard to preconceived mechanistic notions. Fifteen kinases were repeatedly vulnerable in myeloma cells, including AKT1, AK3L1, AURKA, AURKB, CDC2L1, CDK5R2, FES, FLT4, GAK, GRK6, HK1, PKN1, PLK1, SMG1, and TNK2. Whereas several kinases (PLK1, HK1) were equally vulnerable in epithelial cells, others and particularly G protein–coupled receptor kinase, GRK6, appeared selectively vulnerable in myeloma. GRK6 inhibition was lethal to 6 of 7 myeloma tumor lines but was tolerated in 7 of 7 human cell lines. GRK6 exhibits lymphoid-restricted expression, and from coimmunoprecipitation studies we demonstrate that expression in myeloma cells is regulated via direct association with the heat shock protein 90 (HSP90) chaperone. GRK6 silencing causes suppression of signal transducer and activator of transcription 3 (STAT3) phosphorylation associated with reduction in MCL1 levels and phosphorylation, illustrating a potent mechanism for the cytotoxicity of GRK6 inhibition in multiple myeloma (MM) tumor cells. As mice that lack GRK6 are healthy, inhibition of GRK6 represents a uniquely targeted novel therapeutic strategy in human multiple myeloma.