Oleg K. Glebov

Classical and/or alternative NF-κB pathway activation in multiple myeloma

Mutations involving the nuclear factor-kappaB (NF-kappaB) pathway are present in at least 17% of multiple myeloma (MM) tumors and 40% of MM cell lines (MMCLs). These mutations, which are apparent progression events, enable MM tumors to become less dependent on bone marrow signals that activate NF-kappaB. Studies on a panel of 51 MMCLs provide some clarification of the mechanisms through which these mutations act and the significance of classical versus alternative activation of NF-kappaB. First, only one mutation (NFKB2) selectively activates the alternative pathway, whereas several mutations (CYLD, NFKB1, and TACI) selectively activate the classical pathway. However, most mutations affecting NF-kappaB-inducing kinase (NIK) levels (NIK, TRAF2, TRAF3, cIAP1&2, and CD40) activate the alternative but often both pathways. Second, we confirm the critical role of TRAF2 in regulating NIK degradation, whereas TRAF3 enhances but is not essential for cIAP1/2-mediated proteasomal degradation of NIK in MM. Third, using transfection to selectively activate the classical or alternative NF-kappaB pathways, we show virtually identical changes in gene expression in one MMCL, whereas the changes are similar albeit nonidentical in a second MMCL. Our results suggest that MM tumors can achieve increased autonomy from the bone marrow microenvironment by mutations that activate either NF-kappaB pathway.

Characterization of MYC Translocations in Multiple Myeloma Cell Lines

Translocations involving an MYC gene (c >> N >>L) are very late tumor progression events and provide a paradigm for secondary translocations in multiple myeloma. Using a combination of fluorescent in situ hybridization and comparative genomic hybridization arrays (aCGH), we have identified rearrangements of an MYC gene in 40 of 43 independent myeloma cell lines. A majority of MYC translocations involve an Ig locus (IgH > Iglambda >> Igkappa), but the breakpoints only infrequently occur near or within switch regions or V(D)J sequences. Surprisingly, about 40% of MYC translocations do not involve an Ig locus. The MYC translocations mostly are nonreciprocal translocations or insertions, often with the involvement of three chromosomes and sometimes with associated duplication, amplification, inversion, and other associated chromosomal abnormalities. High-density aCGH analyses should facilitate the cloning of MYC breakpoints, enabling the determination of their structures and perhaps elucidating how rearrangements not involving an Ig gene cause dysregulation of an MYC gene.

A rhodium(III) complex for high-affinity DNA base-pair mismatch recognition

A rhodium(III) complex, rac-[Rh(bpy)2phzi]3+ (bpy, 2,2′-bipyridine; phzi, benzo[a]phenazine-5,6-quinone diimine) has been designed as a sterically demanding intercalator targeted to destabilized mismatched sites in double-helical DNA. The complex is readily synthesized by condensation of the phenazine quinone with the corresponding diammine complex. Upon photoactivation, the complex promotes direct strand scission at single-base mismatch sites within the DNA duplex. As with the parent mismatch-specific reagent, [Rh(bpy)2(chrysi)]3+ [chrysene-5,6-quinone diimine (chrysi)], mismatch selectivity depends on the helix destabilization associated with mispairing. Unlike the parent chrysi complex, the phzi analogue binds and cleaves with high affinity and efficiency. The specific binding constants for CA, CC, and CT mismatches within a 31-mer oligonucleotide duplex are 0.3, 1, and 6 × 107 M−1, respectively; site-specific photocleavage is evident at nanomolar concentrations. Moreover, the specificity, defined as the ratio in binding affinities for mispaired vs. well paired sites, is maintained. The increase in affinity is attributed to greater stability in the mismatched site associated with stacking by the heterocyclic aromatic ligand. The high-affinity complex is also applied in the differential cleavage of DNA obtained from cell lines deficient in mismatch repair vs. those proficient in mismatch repair. Agreement is found between photocleavage by the mismatch-specific probes and deficiency in mismatch repair. This mismatch-specific targeting, therefore, offers a potential strategy for new chemotherapeutic design.

DNA mismatch-specific targeting and hypersensitivity of mismatch-repair-deficient cells to bulky rhodium(III) intercalators

Mismatch repair (MMR) is critical to maintaining the integrity of the genome, and deficiencies in MMR are correlated with cancerous transformations. Bulky rhodium intercalators target DNA base mismatches with high specificity. Here we describe the application of bulky rhodium intercalators to inhibit cellular proliferation differentially in MMR-deficient cells compared with cells that are MMR-proficient. Preferential inhibition by the rhodium complexes associated with MMR deficiency is seen both in a human colon cancer cell line and in normal mouse fibroblast cells; the inhibition of cellular proliferation depends strictly on the MMR deficiency of the cell. Furthermore, our assay of cellular proliferation is found to correlate with DNA mismatch targeting by the bulky metallointercalators. It is the Δ-isomer that is active both in targeting base mismatches and in inhibiting DNA synthesis. Additionally, the rhodium intercalators promote strand cleavage at the mismatch site with photoactivation, and we observe that the cellular response is enhanced with photoactivation. Targeting DNA mismatches may therefore provide a cell-selective strategy for chemotherapeutic design.

Gene Expression Patterns Distinguish Colonoscopically Isolated Human Aberrant Crypt Foci from Normal Colonic Mucosa

Aberrant crypt foci (ACF) are considered the earliest identifiable preneoplastic colonic lesions; thus, a greater understanding of the nature of genetic changes underlying the transformation of normal colonic mucosa (NM) into ACF may provide insight into the mechanisms of carcinogenesis. ACF were identified by indigo carmine spraying onto colonic mucosa during colonoscopy and isolated as standard pinch biopsies of the mucosal areas containing the ACF. RNAs isolated from ACF and matched NM biopsies from the ascending and descending colons of 13 patients were analyzed on arrays containing 9128 cDNAs. Thirty-four differentially expressed (P < 0.001) genes were found in a paired comparison of the ACF and NM samples, and 25 of 26 matched pairs of ACF and NM could be correctly classified in leave-one-out cross-validation. Differential expression for seven of eight genes was confirmed by real-time reverse transcription-PCR. Furthermore, ACF and NM samples, including six pairs of ACF and NM samples that had not previously been analyzed by array hybridization, can be correctly classified on the basis of the overexpression in ACF of three selected genes (REG4, SRPN-B5, and TRIM29) evaluated by real-time reverse transcription-PCR. In a separate analysis of 13 biopsy pairs from either ascending or descending colon, ACF and NM samples could also be correctly classified by the gene expression patterns. Analysis of gene expression differences in ACF from the ascending and descending colon versus NM samples indicates that ACF from these distinct colonic locations are converging toward similar gene expression profiles and losing differences in gene expression characteristic of NM from the ascending versus descending colon. (Cancer Epidemiol Biomarkers Prev 2006;15(11):2253–62)

Celecoxib treatment alters the gene expression profile of normal colonic mucosa.

A clinical trial was recently conducted to evaluate the safety and efficacy of a selective inhibitor of cyclooxygenase-2 (celecoxib) in hereditary nonpolyposis colon cancer patients. In a randomized, placebo-controlled phase I/II multicenter trial, hereditary nonpolyposis colon cancer patients and gene carriers received either celecoxib at one of two doses or placebo. The goal was to evaluate the effects of these treatment arms on a number of endoscopic and tissue-based biomarker end points after 12 months of treatment. As part of this trial, we analyzed gene expression by cDNA array technology in normal descending (rectal) colonic mucosa of patients before and after treatment with celecoxib or placebo. We found that treatment of patients with celecoxib at recommended clinical doses (200 and 400 mg p.o. bid), in contrast to treatment with placebo, leads to changes in expression of >1,400 genes in the healthy colon, although in general, the magnitude of changes is <2-fold. Twenty-three of 25 pairs of colon biopsies taken before and after celecoxib treatment can be classified correctly by the pattern of gene expression in a leave-one-out cross-validation. Immune response, particularly T- and B-lymphocyte activation and early steps of inflammatory reaction, cell signaling and cell adhesion, response to stress, transforming growth factor-β signaling, and regulation of apoptosis, are the main biological processes targeted by celecoxib as shown by overrepresentation analysis of the distribution of celecoxib-affected genes across Gene Ontology categories. Analysis of possible cumulative effects of celecoxib-induced changes in gene expression indicates that in healthy colon, celecoxib may suppress the immune response and early steps of inflammation, inhibit formation of focal contacts, and stimulate transforming growth factor-β signaling. (Cancer Epidemiol Biomarkers Prev 2006;15(7):1382–91)

Integrated high-resolution array CGH and SKY analysis of homozygous deletions and other genomic alterations present in malignant mesothelioma cell lines

High-resolution oligonucleotide array comparative genomic hybridization (aCGH) and spectral karyotyping (SKY) were applied to a panel of malignant mesothelioma (MMt) cell lines. SKY has not been applied to MMt before, and complete karyotypes are reported based on the integration of SKY and aCGH results. A whole genome search for homozygous deletions (HDs) produced the largest set of recurrent and non-recurrent HDs for MMt (52 recurrent HDs in 10 genomic regions; 36 non-recurrent HDs). For the first time, LINGO2, RBFOX1/A2BP1, RPL29, DUSP7, and CCSER1/FAM190A were found to be homozygously deleted in MMt, and some of these genes could be new tumor suppressor genes for MMt. Integration of SKY and aCGH data allowed reconstruction of chromosomal rearrangements that led to the formation of HDs. Our data imply that only with acquisition of structural and/or numerical karyotypic instability can MMt cells attain a complete loss of tumor suppressor genes located in 9p21.3, which is the most frequently homozygously deleted region. Tetraploidization is a late event in the karyotypic progression of MMt cells, after HDs in the 9p21.3 region have already been acquired.

Abstract 4208: Integrated oligo aCGH and SKY analysis of genomic alterations in malignant mesothelioma cell lines.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Malignant mesothelioma (MMt) is a rare and very aggressive tumor of the mesothelium. Its most prevalent subtype is malignant pleural mesothelioma, eighty percent of which are associated with asbestos exposure. MMt incidence is 0.9 new cases per 100,000 persons per year. The five year relative survival is 7% overall, which positions MMt immediately above pancreatic cancer (6%), at the bottom of the survival list by cancer site. We applied a comprehensive molecular cytogenetic analysis using spectral karyotyping (SKY) and oligo aCGH to a panel of MMt cell lines. We found the largest set of recurrent and non-recurrent homozygous deletions (HD) for MMt (88 HDs in 17 cell lines; 52 recurrent HDs that fall into ten genomic areas). Detailed analysis of the most frequently homozygously deleted area located at 9p21.3 revealed that the main targets are CDKN2A/p14ARF (in 100% of the MM cell lines), CDKN2A/p16INK4A and CDKN2B /p15 (94% of cell lines). p14ARF seems to be the main target of HDs in the 9p21.3 area (deleted in all 17 MMt cell lines studied), followed by p16INK4A (deleted in 16 cell lines) indicating the inactivation of two major tumor suppressing pathways, p53 and Rb. Accordingly, mutations or HDs of p53 and Rb are infrequent events in MMt tumors. Other recurrent HDs were located at 9p21.2 (targeting LINGO2) and at16p13.3 (RBFOX1) in 41% of cell lines, 22q11.23 (GSTT1) in 29%, 22q12.2 (NF2) and 3q26 (no known genes) in 23% each, 8p11.22 -23 (ADAM5P/ADAM3A) in 18%, 3p21.3-p21.2 (RPL29), 3p21 (DUSP7), 4q22.1 (FAM190A) and 13q11-q12 (LATS2) in 12% each of the 17 MM cell lines While p16INK4A, p14ARF, p15, LATS2 and NF2 have been shown to behave as tumor suppressors in MMts, other genes in the HD areas presented here could be new tumor suppressor candidates. Structural chromosomal rearrangements were mainly non-reciprocal translocations (74% of aberrations detected by SKY) and deletions (23%). No recurrent balanced or unbalanced translocations have been found. Structural chromosomal rearrangements resulted in recurrent losses of fragments or whole chromosomal arms of 1p, 3p, 8p, 9p, 13q, 14q, 15q, 18q, 22q and gains of 1q, 5p, 7p, 8q, 17q, 20q. Small HDs were frequently embedded in the areas of focal losses. Integration of SKY and aCGH data allowed reconstruction of chromosomal rearrangements leading to the formation of HDs. Our data imply that only with acquisition of structural or numerical karyotypic abnormalities can MMt cells attain a complete loss of the 9p21.3 genomic area and loss of tumor suppressor genes located there. Tetraploidization seems to be a late event in the karyotypic progression of MMt cells, after HD in the 9p21.3 area has already been acquired. Citation Format: Ester Rozenblum, Geula Klorin, Oleg Glebov, Robert L. Walker, Yoonsoo Park, Paul S. Meltzer, Ilan R. Kirsch, Frederic J. Kaye, Anna V. Roschke. Integrated oligo aCGH and SKY analysis of genomic alterations in malignant mesothelioma cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4208. doi:10.1158/1538-7445.AM2013-4208