Single-cell template strand sequencing reveals culture-induced chromosomal instability in a gibbon cell line.

PARP inhibitors are emerging as important new anticancer agents, especially in cancers defective in homologous recombination (HR) repair, such as those lacking BRCA1 or BRCA2. To date, the potential role of PARP inhibition in NSCLC has not been extensively studied. As previously reported, we examined a panel of 26 genotypically-defined NSCLC cell lines for sensitivity to the PARP-1 inhibitor AG014699 (rucaparib) using colony formation assays. We identified 5 cell lines that are exquisitely sensitive (IC50 ≈ 50nM), 9 cell lines that are moderately sensitive (IC50 ≈ 60-500nM) and 12 cell lines that are resistant (IC50 > 5 μM). These data were confirmed with a second PARP inhibitor, AZD2281 (Olaparib). To determine whether sensitive cell lines have defective HR compared to resistant cell lines, we examined the formation of RAD51 foci. Both sensitive and resistant cell lines demonstrated efficient formation of RAD51 foci as well as gamma-H2AX foci in response to DNA damaging treatments, indicating correct initiation of HR. In resistant cell lines, the repair process following PARP inhibition is completed within 24 hours, with resolution of RAD51 and gamma-H2AX foci. However, in sensitive cell lines, these foci persist greater than 30 hours following DNA damaging treatments. These results suggest that although sensitive lines are able to initiate HR repair, they are unable to complete the process, so that RAD51 foci are not resolved. The persistence of DNA damage in these cell lines is confirmed by persistent gamma-H2AX focus formation as well as S/G2 arrest, the latter representing intact checkpoint control. Our results suggest that proteins required downstream of RAD51, including helicases and Holliday junction resolvases, are lacking in the sensitive lines. Consistent with this hypothesis, NCI-H520 cells, a highly PARP inhibitor-sensitive cell line, lacks expression of WRN, RECQL4 and RECQL5. siRNA-mediated depletion of these proteins individually sensitizes the resistant A549 cell line to PARP inhibition, with reduction in rucaparib IC50 by 100 fold. Our results may define a novel subset of NSCLCs with a late-step defect in HR that can be approached with PARP inhibition as a novel treatment strategy. Citation Format: Naveen Kommajosyula, Ye Cao, Lisa Moreau, Alan D'Andrea, Geoffrey I. Shapiro. Identification of factors governing sensitivity of NSCLC cell lines to inhibition of Poly (ADP-Ribose) Polymerase (PARP). [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 1787. doi:10.1158/1538-7445.AM2013-1787

A B-Cell like Phenotype Is Associated with Sensitivity to Venetoclax in Multiple Myeloma

Efficacy and Mechanism of Action of the Novel Bromodomain Inhibitor, PLX51107, in B Cell Malignancies

Several indole-3-carbinol analogs are under preclinical evaluation at the National Cancer Institute. One analog, NSC743380 (1-[(3-chlorophenyl)-methyl]-1H-indole-3-carbinol), is selectively toxic to a subset of NCI 60 cell lines in vitro and has undergone extensive in vivo testing. In vivo, NSC743380 produced complete regressions in A498 renal xenograft models at doses as low as 45 mg/kg when administered intraperitoneally. Additional studies demonstrated that NSC743380 is orally bioavailable. To extend knowledge regarding the mechanism of action, two pairs of resistant and sensitive cell lines [A498/ACHN (renal) and NCI-H226/A549 (NSCLC), sensitive/resistant, respectively] were used. Results showed that 5-10 min following NSC743380 treatment, phosphorylation of p38 and JNK were enhanced in sensitive but not resistant lines. Two hrs of treatment resulted in an inhibition of transcription and translation (45% and 75% inhibition, respectively) and by 4 hrs NSC743380 induced caspase-dependent apoptosis with loss of c-FLIP. Pathway-specific inhibitors were then used to gain mechanistic insight. Diverse antioxidants and NSAIDs, inhibitors of JNK, RAS/RAF, PPAR, lipid 2nd messenger signaling, transcription and actin polymerization were shown to completely inhibit NSC743380 activity. Microarray analysis of three sensitive cell lines using Affymetrix U133 Plus 2 chipset identified several trends in the transcriptome notably the upregulation of diverse immediate-early genes (IEGs) including; transcriptional regulators [EGR1, FOS/FOSB, HES1, MAFF and SOX9], secreted factors [HBEGF, IL-8 and GDF-15] and several general IEGs [ARC, ERRFI1, GADD45A/B, GEM and IER2]. A second noteworthy trend involved increased expression of the IL-6 family members, IL-6, IL-11 and LIF. These data led to an exploration as to whether enhanced JAK/STAT signaling was responsible for the above effects. Short time course lysates from 3 sensitive (A498, CAKI-1 and NCI-H226) cell lines and 1 resistant (A549) cell line showed that in sensitive lines only, NSC743380 rapidly (5 minutes) enhanced phosphorylation of the JAK family member Tyk2 at Y1054/55 and STAT3 at Y705. Additionally several phosphostate changes were observed in a subset of sensitive cell lines such as JAK1 phosphorylation in A498 cells and transient increase in pSTAT1 in A498 and CAKI-1 cell lines. Furthermore, two inhibitors of JAK/STAT signaling, AG490 and resveratrol, completely inhibited NSC743380 activity and p38/JNK phosphorylation. Subsequent siRNA knockdown experiments showed that Tyk2 siRNA inhibited NSC743380 activity whereas STAT3 and JAK1 siRNA along with scrambled siRNA had no effect. These data suggest that signaling through the JAK family member Tyk2 contributes towards NSC743380 activity and the data are being evaluated to determine how best to move forward. Funded by NCI Contract No. HHSN261200800001E. 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 2810. doi:1538-7445.AM2012-2810

The numerous and diverse genetic lesions in multiple myeloma (MM) make it difficult to discern those genetic abnormalities that may be suitable therapeutic targets. We have observed that the MYC locus is frequently rearranged in MM (about half of both patients and cell lines), suggesting that MYC dysregulation is a common feature of this disease. To evaluate MYC suppression as a therapeutic strategy in MM, we treated human myeloma cell lines with CPI203, a potent and selective inhibitor of bromodomain and extra-terminal (BET) proteins. The bromodomain inhibitor CPI203 has potency and selectivity similar to that of JQ1, but has improved pharmacokinetics for in vivo studies. From our collection of MM cell lines we have identified sensitive and resistant cell lines and characterized their response to CPI203 in detail. A number of conclusions have emerged from this work. First, response correlates with MYC protein levels after 3 days of treatment. MYC levels are significantly suppressed in sensitive lines and much less affected in resistant lines, supporting the hypothesis that MYC suppression alone determines cell fate in MM cell lines. In sensitive cell lines treatment with CPI203 caused a rapid (within 90 min.) decrease in MYC transcription that is consistent with the response being an intrinsic property of the cell lines. Inhibition of MYC transcription was followed by eventual cell cycle arrest and limited apoptosis in these sensitive cell lines. Both sensitive and resistant cell lines exhibited induction of Histone2H2BE gene in response to CPI203 treatment, indicating that drug efflux pumps alone do not mediate resistance. Surprisingly, several of the resistant cell lines exhibited an initial phase of sensitivity to BET inhibition. Upon drug addition, MYC transcription and MYC protein levels are repressed 2- to 5-fold, followed by a gradual recovery of MYC message and protein levels to the initial levels. In this second phase the cells are truly insensitive to BET inhibition: the drug remains active during this incubation and addition of fresh medium with drug does not affect MYC transcription. Furthermore, many aspects of the BET inhibition gene expression profile remain constant throughout this transition between sensitivity and resistance. These results suggest that the recovery phase is not a simple reversal of BET inhibition but rather the emergence of an alternative-signaling pathway that activates MYC expression in the presence of BET inhibition. Presently we are analyzing gene expression data to identify genes and signaling pathways responsible for this “induced resistance”. The identification of such pathways will help us better understand the molecular basis of the response to BET inhibition, which in turn might enable us to identify predictive biomarkers of the therapeutic response. Citation Format: Daniel L. Riggs, Marta Chesi, P Leif Bergsagel. Targeting MYC in multiple myeloma by BET protein inhibition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5489. doi:10.1158/1538-7445.AM2014-5489

Recently, Poly (ADP Ribose) Polymerase-1 (PARP-1) has been shown to be an important target in subsets of breast and ovarian cancers. PARP inhibitors are emerging as important new anticancer agents, especially in cancers defective in homologous recombination (HR) repair, such as those lacking BRCA1 or BRCA2. To date, the potential role of PARP inhibition in NSCLC has not been extensively studied. BRCA1 or BRCA2 promoter hypermethylation and ATM mutation have been described in primary NSCLCs, alterations that may sensitize cells to PARP inhibition. We have examined a panel of 26 genotypically-defined NSCLC cell lines for sensitivity to the PARP-1 inhibitor AG014699 using colony formation assays. We have identified 5cell lines that are exquisitely sensitive (IC50 ≤ 50nM), 9 cell lines that are moderately sensitive (IC50 ≤ 60-500nM) and 12 cell lines that are resistant (IC50 > 5 µM). To determine whether sensitive cell lines have defective HR compared to resistant cell lines, we examined the formation of Rad51 foci following exposure to PARP inhibition, as well as to gamma-irradiation and cisplatin. Both sensitive and resistant cell lines demonstrated efficient formation of Rad51 foci as well as gamma-H2AX foci in response to DNA damaging treatments, suggesting correct initiation of HR. In resistant cell lines, the repair process following PARP inhibition is completed within 24 hours, with resolution of Rad51 and gamma H2AX foci. No alterations in cell cycle distribution were detected in these cell lines, suggesting that the efficiency of repair allows proliferation to continue largely unabated. However, in sensitive cell lines, these foci persist greater than 30 hours following DNA damaging treatments. These results suggest that although sensitive lines are able to initiate HR repair, they are unable to complete the process, so that Rad51 foci are not resolved. The persistence of DNA damage in these cell lines is confirmed by persistent gamma H2AX focus formation as well as S/G2 arrest, the latter representing intact checkpoint control. We are currently investigating whether PARP inhibitor-sensitive cell lines have a deficiency in one of the helicases or resolvases required for resolution of the complex Holliday junctions formed during HR repair. Our results may define a novel subset of NSCLCs with a late-step defect in HR that can be approached with PARP inhibition as a novel treatment strategy. 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 2540. doi:1538-7445.AM2012-2540

Rationale: ABT-263 (Navitoclax) is one of the BH3 mimetics targeting anti-apoptotic B-cell lymphoma-2 (Bcl-2) family proteins such as Bcl-2, Bcl-XL, and Bcl-w, thereby inducing apoptosis. It has been reported that the response to ABT-263 is associated with expressions of myeloid cell leukemia-1 (Mcl-1), an anti-apoptotic protein. Given its effectiveness as a single agent in preclinical studies, ABT-263 is currently being evaluated in clinical trials for small cell lung cancer (SCLC) and leukemia. However, the efficacy of ABT-263 in non-small cell lung cancer (NSCLC) has not been fully evaluated. We examined the effect of ABT-263 on cell proliferation of NSCLC cell lines and investigated the underlying mechanisms. Methods: The following 9 NSCLC cell lines were examined: SK-LU-1, A549, H358, Calu3, H3122, H1975, H460, H441, and BID007. The effects of ABT-263 in NSCLC cell lines were evaluated by MTS assay. Apoptosis was examined by flowcytometry using staining for annexin V and propidium iodide (PI), and also western blotting for cleaved PARP. Quantitative RT-PCR was carried out to assess the mRNA expression levels of anti-apoptotic genes and pro-apoptotic genes. Immunoprecipitation and western blotting were performed to compare the levels of anti-apoptotic and pro-apoptotic proteins between the sensitive and resistant cell lines. In addition, knockdown of Mcl-1 was performed by siRNA. Results: By screening 9 NSCLC cell lines using MTS assay, we found Calu3 and BID007were sensitive to ABT-263. We also confirmed that apoptosis was induced only in the ABT-263 sensitive lines but not in the ABT-263 resistant cell lines after ABT-263 treatment. However, the expression levels of Bcl-2 family proteins, including Mcl-1, did not differ significantly among the ABT-263 sensitive and resistant cell lines. Unlike the results in previous reports regarding SCLC, Mcl-1 was not decreased in the sensitive cell lines. The ABT-263 resistant cell lines became sensitive to ABT-263 after knockdown of Mcl-1 by siRNA, while the ABT-263 sensitive cell lines maintained the same sensitivity. Conclusion: We found that Calu3 and BID007 were sensitive to ABT-263. In the sensitive NSCLC cell lines, ABT-263 induces apoptosis irrespective of Mcl-1 expression levels. Citation Format: Aoi Kuroda, Keiko Ohgino, Hiroyuki Yasuda, Junko Hamamoto, Daisuke Arai, Kota Ishioka, Tetsuo Tani, Shigenari Nukaga, Ichiro Kawada, Katsuhiko Naoki, Kenzo Soejima, Tomoko Betsuyaku. ABT-263 is effective in a subset of non-small cell lung cancer cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4. doi:10.1158/1538-7445.AM2015-4

14618 Background: Lapatinib is a HER1 and 2 tyrosine kinase inhibitor (TKI). Little is know about its interaction with other chemotherpy drugs. In this work investigate interactions between lapatinib and cisplatin or SN-38 (the active metabolite of irinotecan) in cisplatin sensitive (testis cancer) and resistant cancer cell lines. Methods: We obtained 3 cancer cells lines (833K, GCT27, SUSA) all of which had cisplatin resistant daughter lines (833Kr, GCT27r, SUSAr). These resistant lines were established by exposing the sensitive lines to increasing doses of cisplatin. Western Blot analysis was used to estimate HER 1–4 levels and expression of proteins in the PI3K and MAPK pathways. The IC 50s for lapatinib, cisplatin and SN-38 were calculated in these cell lines. These drugs were then given in combination, and the effect was estimated using calcusin plots. Flow cytomery was used to estimate the effect on the cell cycle and apoptosis. Identical experiments were carried out with Pazopanib, instead of lapatinib, which was used as a control. Results: The IC50 for lapatinib in the resistant and sensitive cell lines were similar (4.0–5.0 micromolar and 3.6–4.4 micromolar respectively). Sensitivity was not related to HER 1–4 expression. Synergistic interactions were observed between SN-38 and lapatinib in all 6 cell lines (calculated combination indices <0.7 in all lines). No synergistic interaction was seen between cisplatin and lapatinib. Cell cycle data revealed the synergistic combination was associated with an increase in apoptosis and reduces cell cycle arrest. Pazopanib was not associated with a synergistic interaction in any of the cell lines with either cisplatin or SN-38. Conclusions: Lapatinib appears equally efficacous in both cisplatin sensitive and resistant cell lines. A synergistic interaction occurs between lapatinib and SN-38 in this model which is associated with increased apoptosis. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration GlaxoSmithKline

The role of cytogenetic analysis in the diagnosis and management of haematological malignancies is undisputed.The accuracy of cytogenetic diagnosis has improved steadily over the past 20 years, primarily due to a series of technical developments. However, despite improvements in high-resolution banding and culture methods to detect the chromosomally abnormal cells, many haematological malignancies are retractable to conventional cytogenetic analysis. This may be due to the presence of multiple abnormal clones, complex rearrangements, a low mitotic index, or poor chromosome morphology. Since the late 1980s a range of techniques based around fluorescence in situ hybridization (FISH) have greatly enhanced cytogenetic analysis. These use a variety of nucleic acid sequences as probes to cellular DNA targets and serve to bridge the gap between molecular genetic and conventional cytogenetic methods. Virtually any genomic DNA can now be used as a probe with which to investigate a wide variety of DNA targets, from metaphase chromosomes to mechanically stretched DNA fibres. The simultaneous detection of multiple target regions is also possible, using differentially labelled probes detected by different colours. In research, FISH has played a pivotal role in the identification of non-random translocations and deletions, pinpointing regions which contain genes involved in leukaemogenesis. Now, at the cutting edge, a new set of resources and technical innovations herald a new era for molecular cytogenetics, with colour karyotyping, comparative genomic hybridization (CGH) microarrays and mutation detection using padlock probes providing the promise of the future. The number of applications for FISH is almost unlimited (see Table I for some pertinent examples). This review will concentrate on the most recent developments in FISH which have had a considerable impact on the cytogenetic diagnosis and study of haematological malignancies, with some insight into the possible future roles for this flexible technology. The application of FISH to metaphase chromosomes provides unequivocal evidence of chromosome rearrangements. There are many different types of cloned or uncloned DNA which can be used for as a probe for FISH (reviewed in Buckle & Kearney, 1994). However, the most commonly used probes in cytogenetic analysis of haematological malignancy are: (i) repetitive sequence centromeric probes, (ii) whole chromosome paints, and (iii) locus-specific probes. Chromosome-specific centromeric probes which target tandemly repeated alpha (or beta) satellite sequences present in the heterochromatin of the chromosome centromeres are used to detect numerical chromosome abnormalities. Centromeric probes are commercially available for all human chromosomes and these provide a rapid and simple way of enumerating specific chromosome pairs, both in metaphase and interphase. This type of analysis is useful in many types of leukaemia where the chromosome morphology is poor and banding indistinct, such as in hyperdiploid acute lymphoblastic leukaemia (ALL). However, centromeric probes only give information on the number of centromeres of a particular type present; they cannot tell whether the chromosome is structurally abnormal. Whole chromosome painting probes are complex mixtures of sequences from the entire length of a specific chromosome. These are also available for all human chromosomes, and can be used to delineate chromosome pairs (Cremer et al, 1988; Pinkel et al, 1988). Whole chromosome painting probes (paints) can be derived from chromosome-specific libraries, PCR amplification of flow-sorted chromosome fractions, or microdissected DNA specific for each chromosome (Collins et al, 1991; Telenius et al, 1992; Vooijs et al, 1993; Guan et al, 1996). Chromosome paints are most useful for identifying the components of highly rearranged and marker chromosomes, where the banding pattern cannot be relied upon. However, their usefulness is limited to metaphase analysis, as the extended chromosome domains in interphase are often diffuse and difficult to quantitate. In addition, chromosome painting is a relatively insensitive technique and cannot detect small interstitial deletions, duplications or inversions. The resolution for the detection of small telomeric translocations is also limited. Single locus probes detect specific sequences present in only one copy. When using these probes the efficiency of hybridization needs to be considered; the larger the target sequence the more efficient the hybridization. Single-copy probes cloned in cosmid, YAC, P1, PAC and BAC vectors all give reliable FISH signals, with a fluorescent signal on both chromosome homologues in >90% of metaphases. Structural rearrangements detected using this type of probe include translocations, inversions and specific deletions (Dauwerse et al, 1990; Tkachuk et al, 1992; Sacchi et al, 1995; Jaju et al, 1998). The use of specific gene probes for chromosomal translocations has simplified the process of identifying known translocations, especially in complex or masked versions of the translocation (e.g. BCR/ABL, PML/RARα fusions), and has particular applications for interphase analysis. One of the greatest advances in cytogenetic analysis facilitated by FISH has been the ability to use non-dividing cells as DNA targets, referred to as interphase FISH (Cremer et al, 1986). This enables the screening of large numbers of cells and provides access to a variety of sources of haemopoietic cells including blood and bone marrow smears and haemopoietic progenitor cells from colony assays (Bentz et al, 1993; Poddighe et al, 1993; Mühlmann et al, 1998). This has considerable advantages for some haemopoietic malignancies, where the proliferative activity is low, or when the mitotic cells do not represent the neoplastic clone, for example chronic lymphoblastic leukaemia (CLL), Hodgkin's disease, multiple myeloma. Interphase FISH permits the identification of both numerical and structural chromosome abnormalities both as an aid to cyto-genetic diagnosis and for monitoring disease progression. Interphase FISH has had a major impact on the cytogenetic analysis of B-CLL, revealing a much higher incidence of trisomy 12 than found by conventional cytogenetic analysis (Anastasi et al, 1992; Garcia-Marco et al, 1997). An examination of the relationship between clinical stage and trisomy 12 showed an association with atypical morphology, advanced stage of disease and low proliferative activity. In addition, immunophenotyping and FISH showed that the +12 is present in only a proportion of clonal B cells (Garcia-Marco et al, 1997). All of this data suggests that trisomy 12 is a secondary event in the development of CLL. For chromosome deletions, specific locus or region-specific probes have been used to demonstrate a high frequency of mono-allelic deletions of the RB1 and p53 genes in B-cell malignancies (Stilgenbauer et al, 1993, 1995; Döhner et al, 1995; Cano et al, 1996). Interphase FISH was also instrumental in identifying the critical region of deletion on 11q13 associated with B-cell lymphoid malignancy, which consequently identified mutations of the ATM gene in T-prolymphocytic leukaemia (PLL) (Stilgenbauer et al, 1997). DNA probes for the fusion genes involved most specific chromosomal translocations and inversions in leukaemia are now commercially available. The differential labelling and detection of these probes in different colours enables a direct visualization of the fusion gene. The simplest scheme is to use two probes (one from each of the fusion genes), differentially labelled and detected with two different-coloured fluorochromes (see Fig 1A). An interphase cell positive for the translocation will exhibit a red–green fusion signal representing the translocation, and a single red and green signal corresponding to the normal chromosome homologues. However, the false positive rate using this approach is quite high (approximately 5%). In addition, the presence of variant translocations or translocations in which the breakpoints are spread over a large distance (e.g. Burkitt's lymphoma), means that the false negative rate can also be quite high. There are several more complex strategies to overcome this (see Figs 1B and 1C). Firstly, if a series of probes spanning both translocation breakpoints are used, this will result in splitting of both fluorescent signals, and the presence of two red–green fusions. Another, more complex, strategy is to employ three or even four different colours, so that the incidence of false positives and false negatives is reduced (Ried et al, 1993; Sinclair et al, 1997). However, the more complicated the colour scheme, the more difficult and complex the analysis. At present, this analysis is done manually, so this is a serious consideration. . Schematic representation of the detection, by FISH, of the Philadelphia translocation in interphase nuclei. In each case the left-hand panel shows the location of the FISH signals on metaphase chromosomes (partial karyotype), and the right-hand panel the interphase FISH signals. In (A) two probes from the flanking regions of the BCR and ABL genes are labelled and detected in different colours: BCR in red and ABL in green. The BCR/ABL fusion results in co-localization of the red and green signals on the der(22) (Philadelphia) chromosome, with a single red and green signal separated, corresponding to the normal chromosomes 22 and 9, respectively. A BCR/ABL negative cell would show two separate red and two green signals. The scheme in (B) uses two probes, this time spanning both the BCR and ABL breakpoint regions. In this case, two red/green fusion signals are formed: one corresponding to the der(9), and the second to the der(22). A positive cell would therefore exhibit one red, one green and two red/green fusions (from Dewald et al, 1998). In (C), a third probe from the region just proximal to ABL on 9q34 is used, labelled in a different colour (represented here in yellow). A translocation positive cell exhibits one green/yellow doublet, one red/green and a single red and yellow signal (from Sinclair et al, 1997). The possibility of using interphase FISH as screening test for specific abnormalities found in acute myeloid leukaemia (AML) subtypes was recently described by Fischer et al (1996). This study used 23 different probes and six to eight hybridizations per patient. They found that interphase FISH was more sensitive for the detection of t(8;21), inv(16), +8q, +11q, +21q, +22q and −Y, and obtained a cytogenetic result in a proportion of cases with no evaluable metaphases. However, this kind of analysis may eventually be replaced by disease-specific DNA chips (see Matrix-CGH below). The detection of residual Philadelphia-positive cells is important after allogeneic bone marrow transplant or interferon (IFN) treatment. In particular, the degree of response to IFN treatment has been shown to be an independent prognostic indicator. The sensitivity of conventional cytogenetics is around 5%, and may be difficult due to low mitotic rate of cells after treatment. RT-PCR is the most sensitive method for detection of BCR-ABL (approximately 10−6) but quantification is difficult. Interphase FISH offers the prospect of using peripheral blood samples, reducing the need for frequent bone marrow aspirates. However, 'in house' cut-off levels must be established for each probe set. Conventional FISH probes for the detection of BCR-ABL gene fusion in interphase cells have suffered from a high false positive rate (Tkachuk et al, 1992). The development of three-colour/three-probe FISH protocols for BCR-ABL detection has significantly lowered the false positive rate, and also increased the sensitivity of detection (Sinclair et al, 1997; Dewald et al, 1998). Sinclair et al (1997) used a third probe (for the ASS gene) 200 kb proximal to ABL, such that when a true BCR-ABL fusion was present, there was one co-localization for BCR-ABL, and a separate ASS signal corresponding to the der(9). In cells where the BCR and ABL signals co-localized due to chance, the ASS signal co-localized with the red ABL signal on both chromosomes 9 (see Fig 1C). This three-colour approach resulted in a low false positive and false negative rate. Dewald et al (1998) used a similar strategy, with probes spanning both the BCR and ABL breakpoints. This resulted in two different co-localizations: one representing the der(22) and the other the der(9) chromosomes (see Fig 1B). Strict scoring criteria, experienced operators and scoring of >3000 cells all enabled the detection of residual disease in 0.079% of cells. This skilled and time-consuming approach was also successful in detecting variant translocations. Although the sensitivity of dual-colour interphase FISH is less than for RT-PCR, PCR is not a possibility in a number of cases, for example for the detection of deletions, monosomy or trisomy. Interphase FISH has been used for the detection of residual disease after allogeneic bone marrow transplantation (Anastasi et al, 1991; Wessman et al, 1993). Kasprzyk & Secker-Walker (1997) studied hyperdiploid karyotypes in ALL to detect minimal residual disease. Using three-colour interphase FISH, targeting three chromosomes simultaneously, they were able to achieve a sensitivity of 10−4, and predict relapse in a number of cases. The ability to combine interphase FISH analysis with immunological staining for cell surface antigens provides a powerful method to combine cell by cell analysis with morphology or immunophenotype. Simultaneous immunophenotyping and FISH analysis has been used to investigate lineage involvement in myelodysplastic syndrome (MDS), chronic myeloid leukaemia (CML) and other myeloproliferative syndromes (Price et al, 1992; Nylund et al, 1993; Torlakovic et al, 1994; Soenen et al, 1995; Haferlach et al, 1997, reviewed in Knuutila, 1997). Concurrent immunophenotype and FISH analysis has also been used to demonstrate that the leukaemia which emerged 5 years after sex-mismatched allogeneic bone marrow transplant occurred in donor cells (Katz et al, 1993). In CML, three-colour detection of the Philadelphia translocation and immunophenotype enabled the identification of the translocation in CD20-positive B cells (Torlakovic et al, 1994) and more recently CD3-positive T cells and CD34-positive precursor cells (Haferlach et al, 1997). This supports the belief that CML is a disorder of an early progenitor cell, capable of differentiating into myeloid and some lymphoid lineages (reviewed in Knuutila, 1997). There are also reports of the clonal involvement of B cells in MDS, using del(20q) and monosomy 7 as clonal markers (White et al, 1994; van Lom et al, 1995). In Hodgkin's disease the low percentage of Hodgkin and Reed-Sternberg (HRS) cells means that even interphase FISH may not detect clonal abnormalities. In a recent study the combination of CD30+ staining and FISH with pairs of centromeric probes revealed numerical abnormalities in 100% of HRS cells (Weber-Matthiesen et al, 1995). Surprisingly, clonal abnormalities found in metaphase analysis were not consistent with the interphase FISH analysis, indicating that metaphase analysis of Hodgkin's disease may not be informative. FISH has proved an invaluable aid in the mapping of translocation breakpoints, resulting in the identification of many fusion genes (reviewed in Rabbitts, 1994). A recent addition to the repertoire of FISH techniques now provides significant advantages over other molecular methods for mapping breakpoints which are dispersed over large distances. The term Fibre-FISH is used to describe a collection of methods for performing FISH to extended DNA stretched out on a glass slide (Wiegant et al, 1992; Parra & Windle, 1993; Bensimon et al, 1994; reviewed in Raap, 1998). vandraager et al (1996) have demonstrated the usefulness of this technique for mapping breakpoints of the cyclin D1 gene in mantle cell lymphomas. Using a series of overlapping probes from the 11q13 breakpoint region labelled in alternating red and green fluorochromes creates a colour bar code for the region. Translocations are recognized by the disruption of this bar code into its two complementary parts. The advantages of this method over Southern blotting or pulsed field gel electrophoresis are its simplicity and speed: only a few images need to be examined, and chromosomal breaks over a distance of 250 kb can be visualized. However, the parameters underlying the technique are poorly understood, and at present it remains a research rather than diagnostic tool, confined to a few specialist laboratories. The strength of conventional (G-banded) cytogenetic analysis has always been the ability to survey the entire genome for clues to pathogenesis. However, the poor chromosome morphology and low mitotic index of many leukaemias and lymphomas means that conventional cytogenetic analysis is often limited. In addition, the analysis of banding pattern in highly rearranged karyotypes is difficult and unreliable. One of the remaining challenges for the new FISH techniques is to identify cryptic rearrangements, particularly involving telomeric regions, in apparently normal karyotypes. A significant proportion (15–20%) of bone marrow karyotypes in leukaemia are reported as normal by conventional (G-banded) cytogenetic analysis. Despite significant improvements in the quality of leukaemic metaphase preparations over the past decade, the abnormality rate has not improved. The t(12;21)(p13;q22) remained undetected until 1994, despite the fact that it accounts for 25% of childhood B-cell ALL cases (Romana et al, 1994). This translocation still remains undetectable by conventional cytogenetic analysis. The difficulty in detecting chromosome abnormalities such as this in the fact that there is a of staining regions of a similar The recent development of whole chromosome painting provides the promise of identifying cryptic chromosome rearrangements, a of all chromosome abnormalities in a single FISH using the method of probe labelling was described by et al In this probes are labelled with mixtures of fluorochromes such that no two probes have the The number of targets which can be in this is where number of fluorochromes available. FISH with to different colours has been available for a number of years, using probes labelled with three fluorochromes (Dauwerse et al, 1992; et al, 1992). the number of fluorochromes to enables the identification of all pairs of human The has been due in to the of new fluorochromes in the and and to two detection methods to mixtures of fluorochromes et al, et al, 1996). of these used a set of whole chromosome paints, labelled with different mixtures of The detection FISH relied on separate images for each of using et al, 1996). The labelling combination for each chromosome was and in using The second used a single of the and a combination of and et al, 1996). An was used to the at each of the of these techniques have demonstrated chromosome rearrangements in complex karyotypes in cell and in haematological malignancies et al, et al, 1997; also Fig However, the sensitivity of both or remains to be The of this are the on metaphase analysis, and the resolution of painting probes. All of the available whole chromosome paints are in some of the particularly the telomeric regions. that the sensitivity of painting for the detection of translocations involving regions may be as low as also Fig In addition, whole chromosome painting will not detect deletions, duplications or inversions. In both and still to the and a combination of FISH is still to identify all abnormalities in complex karyotypes. . FISH to the analysis of a complex in the myeloid cell (A) after analysis. The structural abnormalities identified are: A cryptic was also present, but difficult to identify by analysis. (B) A metaphase after analysis. of amplification are in green and deletions in of the genome which are regions were identified the entire chromosome of and A deletion of due to the of an The analysis identified the of a marker chromosome, as as revealing several cryptic translocations in The of the analysis was to the of the with large deletions translocations in most cases. et al (1997) have recently described an approach which use of the regions of between different to approach a of colour of The of a colour for each chromosome was described by et al using a series of from the length of the chromosome, labelled differentially and detected in a different banding on the of between and has been useful in comparative of regions (reviewed in et al, 1997). The by and have now a set of paints derived from cell Chromosome-specific painting probes were derived from and by chromosome and When used for FISH to human metaphase chromosomes, this resulted in the of each chromosome into between two and six labelling using three fluorochromes resulted in a colour banding pattern for each chromosome. In with specific an colour can be Although at present the number of colour is the of this approach is with the to identify chromosomal inversions and colour banding has been used to identify cryptic translocations in CML A set of chromosome-specific probes which identify the of all human chromosomes the of the is now available for FISH of and of 1996). These contain DNA sequences cloned in cosmid, and PAC clones, the of which have been to between and kb from human chromosome These probes have been in a FISH for rearrangements on a series of with cryptic chromosome rearrangements et al, 1997). This is dual-colour an of all chromosome regions on a single However, the approach a high mitotic index and is most for the analysis of which on peripheral blood or where a cell is available. In the of these probes for leukaemic karyotypes has been to identify the specific region in rearrangements found by painting (see Fig An a FISH would the of all chromosome regions in a single However, the of labelling and multiple colour detection methods for cosmid, or even and PAC a series of developments. Firstly, the simultaneous analysis of all chromosome in a different colour the number of targets with fluorochromes is the of such an would on it is not whether the targets of such small probes labelled with several different fluorochromes can be due to of resolution of the is that the development of fluorochromes will this type of analysis . The use of chromosome-specific probes to identify the of chromosome on two In each case dual-colour hybridization was out with the probe labelled in and detected in red fluorescent and the probe labelled with and detected with fluorescent In (A) probes for and identified the on the as derived from In (B) the on was identified as from Although these abnormalities were detected by painting no information of the chromosomal region is also important to that the abnormality in (A) was described by as The of all of the new is that they still metaphase The advantages of are that it the need for cells and not any of the chromosome The is genomic and DNA are labelled with different in and normal metaphase The in number between the normal and is by in red and green fluorescence the length of the chromosome (see Fig the 5 years its et al, has a of identifying new regions of amplification and deletion in a wide variety of types (reviewed in et al, 1997). The use of for haematological malignancies is more limited (Bentz et al, et al, et al, et al, 1997). The of for haematological malignancies are the to detect rearrangements, and the for cells with the clonal However, and some lymphomas have from the application of (Bentz et al, et al, et al, 1996). One study of identified and not identified or not detected by clonal were identified in six out of cases with a normal (Bentz et al, The for results between and were a complex the or a of metaphases. This study that banding analysis may abnormalities and may important chromosome have not been identified in CLL. In a study of myeloid leukaemias found a between and results (Bentz et al, The only were a of to detect and The major of is its due to the on metaphase For deletions, the resolution of has been at (Bentz et al, 1998). the most future for in to cloned DNA (see below). This to overcome the of using metaphase chromosomes as a target for by metaphase chromosomes with cloned DNA in small and to the surface of a glass et al (1997) used for the detection of high number amplification using as For low number larger cloned probes or were For deletions, a resolution of it not between and The other of metaphase also at of clonal cells and will not detect translocations. One types of (i) disease-specific probe (ii) or (iii) DNA for specific regions, at over the whole probes are DNA in which the has been replaced by The rapid and of with complementary DNA sequences for a number of including FISH probes have been for the human telomeric the fluorescent detection of all in a single These signals to be a for fluorescence than conventional FISH signals, an of length et al, 1996). This may also be extended to other sequences such as centromeric may also be to combine telomeric probes with the chromosome-specific DNA probes to provide a of and specific chromosome This new the promise of detecting single in cells. probes of two different each 20 by a When the probe sequence the the and of the probe are and the probe is et al (1997) used two different probes, each labelled with a different to detect single in a centromeric The sensitivity of this may be improved by the of new sensitive labelling techniques such as the use of fluorescent signal et al, 1995; et al, 1995). to the sensitivity is amplification of the This to the would fluorescent detection of mutations in nuclei. amplification has been with some using extended DNA from but at this stage not or on et al, 1998). In the relatively time its FISH has had a major impact on cytogenetic analysis, due to the sensitivity and of its Although some of the applications will research the and probes for most cytogenetic abnormalities are now the of most clinical laboratories. However, conventional FISH can only provide to the specific and some of the The recent of FISH to the visualization of the entire human genome in different colours has the of and The of this approach is the ability to the whole genome in a single hybridization the screening of cytogenetics with the accuracy of molecular The belief that cytogenetics is more an than a has been from the the aid of new colour techniques and cytogenetic analysis now a molecular of The major impact of this development in field of haematological is to be the identification of new and non-random chromosome rearrangements and clinical of the most recent innovations to The most of fluorescent metaphase is now by and and will not only the of such but the sensitivity of interphase FISH analysis. many of the of cytogenetic analysis by the future for cytogenetics has The all of the of the particularly for the and analysis of the cell of the described here was by the and the

The histone deacetylase inhibitor (HDACi) sodium butyrate promotes differentiation of colon cancer cells as evidenced by induced expression and enzyme activity of the differentiation marker intestinal alkaline phosphatase (ALPi). Screening of a panel of 33 colon cancer cell lines identified cell lines sensitive (42%) and resistant (58%) to butyrate induction of ALP activity. This differential sensitivity was similarly evident following treatment with the structurally distinct HDACi, MS-275. Resistant cell lines were significantly enriched for those harboring the CpG island methylator phenotype (p = 0.036, Chi square test), and resistant cell lines harbored methylation of the ALPi promoter, particularly of a CpG site within a critical KLF/Sp regulatory element required for butyrate induction of ALPi promoter activity. However, butyrate induction of an exogenous ALPi promoter-reporter paralleled up-regulation of endogenous ALPi expression across the cell lines, suggesting the presence or absence of a key transcriptional regulator is the major determinant of ALPi induction. Through microarray profiling of sensitive and resistant cell lines, we identified KLF5 to be both basally more highly expressed as well as preferentially induced by butyrate in sensitive cell lines. KLF5 overexpression induced ALPi promoter-reporter activity in resistant cell lines, KLF5 knockdown attenuated butyrate induction of ALPi expression in sensitive lines, and butyrate selectively enhanced KLF5 binding to the ALPi promoter in sensitive cells. These findings demonstrate that butyrate induction of the cell differentiation marker ALPi is mediated through KLF5 and identifies subsets of colon cancer cell lines responsive and refractory to this effect.

Background: NHWD870 (Ningbo Wenda Pharma, China) is a novel oral bromodomain and extraterminal (BET) protein family inhibitor. BET proteins are transcriptional co-activators that interact with multiple co-regulatory molecules at gene promoters and enhancers. BET inhibitors disrupt transcriptional regulatory complexes and have broad anticancer activity. The relation between the cellular pharmacokinetics (PK) and targeted-gene regulation governing the biological effects of BET inhibitors is still largely not understood. We analyzed cellular PK properties and CMYC and related genes modulation in NHWD870-sensitive and resistant leukemic cell lines. Materials and Methods. Established human cell lines from acute and chronic myeloid leukemia (HL-60, U937, K562), acute lymphoblastic leukemia (Jurkat, MOLT-3) and SET-2 a JAK2 V617F mutated-megakaryoblastic cell line were studied. Anti-proliferative effects of NHWD870 were assessed by MTT after 72h-exposure. For cellular PK studies, cells were seeded at 2.106cells/ml and exposed to 245.5 ng/ml (500nM) NHWD870 for 0, 3, 10, 30, 60, 120 and 180 min. At each time point, NHWD870 extracellular and intracellular concentrations were analyzed in cell supernatants and pellets respectively, using Ultra Performance Liquid Chromatography with tandem Mass Spectrometry (concentration range 0.1-100 ng/mL). In cell pellets, protein modulation was analyzed by Western blot using commercial antibodies. Results: IC50 values were between 20-600 nM in HL60, U937, SET-2 and Jurkat cells and were ≥ 1,000 nM for other cell lines. Cellular uptake of NHWD870 was rapid (3 min) in both sensitive and resistant leukemic cell lines, with a mean concentration of ~5.0 ng/106 cells (3-8.5 ng/106cells) and ~3.1 ng/106 cells (2.8-3.5 ng /106cells) for HL60 and K562 cell line respectively, whereas extracellular levels of NHWD870 were stable with up to 3h exposure. In the resistant cell line, K562, we observed CMYC mRNA downregulation after 2h exposure, even though NHWD870 intracellular levels were detected after 5 min exposure. A rapid downregulation of CMYC and P21 proteins were observed in the sensitive cell lines, HL60, U937, SET-2 and Jurkat, whereas other BET-targeted genes, such as HEXIM and BCL2 levels were modulated after longer exposure time points in sensitive cell lines. Conclusions: NHWD870 intracellular accumulation occurred to a similar extent and timing in NHWD870 sensitive and resistant leukemic cell lines. Rapid modulation of NHWD870-target genes was observed at the protein levels only in sensitive cell lines. Citation Format: Maria E. Riveiro, Samuel Huguet, Marie-francoise Gauzan, Mohamed Bekradda, Nenghui Wang, François Lokiec, Olivier Madar, Keyvan Rezai. Cellular pharmacokinetics and molecular pharmacodynamics studies of a novel BET inhibitor NHWD870 in sensitive and resistant leukemic cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 801.

The Effect of Statins in Colorectal Cancer Is Mediated Through the Bone Morphogenetic Protein Pathway

The role of glutathione (GSH) and GSH-S-transferase (GST) activity in modulating the cytotoxicity of four platinum drugs and melphalan was evaluated in eight human ovarian carcinoma cell lines. The cell lines were established from solid and ascitic tumours from pretreated and untreated patients, and showed a wide spectrum of sensitivity to several platinum II and platinum IV drugs; cisplatin, carboplatin, CHIP and tetraplatin. Intracellular glutathione concentration measured in the cell lines showed a significant (P = 0.05) correlation with IC50 values for cisplatin (r = 0.91), carboplatin (r = 0.87) and CHIP (r = 0.88). The correlation between GSH levels and IC50 values for melphalan (r = 0.76) or tetraplatin (r = 0.60) was not as significant. GST activity showed no correlation with IC50 values, for the four platinum drugs. To determine the significance of the elevated GSH concentration in the refractory cell lines, the effect of D,L-buthionine-S, R-sulfoximine (BSO) mediated GSH depletion on platinum drug cytotoxicity was examined in one of the most sensitive (CH1) and two of the least sensitive (relatively resistant; SKOV-3, HX/62) cell lines. Comparison was made with the effect of GSH depletion on melphalan cytotoxicity in these three lines. These lines were differentially sensitive to BSO, with the two most platinum drug resistant lines being more tolerant to BSO than the sensitive CH1 line. Depletion of cellular GSH, ranging between 61 and 88%, had a differential effect on the sensitivity to PtII vs PtIV drugs in the three cell lines: cytotoxicity of the PtIV drugs, tetraplatin and CHIP, was substantially enhanced in both the resistant and sensitive cell lines; in contrast, the cytotoxicity of the PtII drugs, cisplatin and carboplatin, was only significantly increased in one of the two relatively resistant lines (SKOV-3) and in the sensitive (CH1) line after GSH depletion. Moreover the dose modification factor (DMF) for the PtII agents were lower than those for PtIV agents in the three cell lines. The dose modification factor for tetraplatin after BSO treatment was similar to that observed for melphalan in all three cell lines. In the SKOV-3 cell line extending the BSO pretreatment period to 48 h from 24 h marginally reduced the cytotoxicity of cisplatin, whereas the cytotoxicity of the other three drugs remained similar to that observed after 24 h BSO pretreatment. In contrast, extending the BSO treatment to 24 h after drug exposure potentiated the cytotoxicity of cisplatin, CHIP and tetraplatin.(ABSTRACT TRUNCATED AT 400 WORDS)

In Silico Prediction of Novel Drug Combinations to Combat Bortezomib-Resistant Multiple Myeloma

7533 The efficacy of anthrax tumor toxin (LeTx) was tested on a panel of 18 human melanoma cell lines using a 3H-Leucine incorporation inhibition assay. 11/18 cell lines were sensitive to LeTx (IC50 < 350 pM, percent cell kill > 75%), while the remaining 7/18 were not sensitive to LeTx (IC50 > 750 pM, Percent cell kill < 60%). 10/11 sensitive melanoma cell lines carried the V599E BRAF. Only 1/7 resistant cell line was BRAF mutation positive (p < 0.0001). 6/7 resistant cell lines carried the Q61R or Q61K N-Ras mutation. Next, LeTx effects on 3H-Thymidine incorporation were measured. In 11/11 sensitive cell lines, the IC50 for 3H-thymidine incorporation was lower than that of 3H-Leucine incorporation indicating that the majority of surviving cells at maximal concentration were in cell cycle arrest. 4/7 cell lines that were not sensitive to LeTx by 3H-Leucine incorporation had low IC50 values in the 3H-Thymidine incorporation inhibition assay (IC50 < 275 pM) and a significantly lower percentage of 3H-thymidine uptake at maximum concentration suggesting significant tumor cell growth arrest rather than tumor cell death. Anthrax toxin receptor (ATR) levels, measured by saturation assay with 125I-PA, varied between 3562 and 32440 receptors/cell with resistant cell lines having the same levels of ATR expression as sensitive cell lines and no correlation of ATR receptor levels with IC50 for LeTx sensitive cell lines. Total and phosphorylated levels of MEK1/2 and ERK1/2 in the cell lines were quantified by western blots and densitometry. LeTx killing correlated with MEK1/2 activation levels. 9/11 sensitive melanoma cell lines and 2/7 resistant melanoma cell lines had a P-MEK1/2 to MEK1/2 ratio > 0.3 (p < 0.001). P-ERK1/2 to ERK1/2 levels did not correlate with melanoma cell line sensitivity to LeTx. The small molecular weight MEK1/2 inhibitor U0126 was tested for effects on the cell lines. 6/6 LeTx resistant cells were resistant to U0126; 5/9 LeTx sensitive cells were sensitive to U0126. In summary, melanoma cell line LeTx sensitivity correlated with B-Raf mutation, P-MEK1/2 levels, and U0126 sensitivity, but not ATR or P-ERK1/2. In future clinical trials of LeTx in metastatic melanoma, these results may provide molecular targets to identify responders. No significant financial relationships to disclose.