Evidence for ABL Amplication in Multiple Myeloma and Its Role in Treatment

Background: The cytogenetic abnormalities are considered as initiating events in the pathogenesis of multiple myeloma (MM) and were assumed to be clinical signicance. A number of dened cytogenetic lesions have been reported by genetic analysis techniques, while ABL gene, known as the therapeutic target in chronic myelogenous leukemia (CML), its expression in myeloma has not been deeply explored. Methods: We used publically available method FISH to analyze the chromosomal architecture, clinic features and overall survival of 101 MM patient samples. Additionally, we examined ABL expression in MM cell lines (NCI-H929, LP-1 and U266) through FISH and western blot. After culturing with ABL kinase inhibitor STI571, we analyzed MM cell proliferation by CCK8 assay and detected ABL protein levels by western blot. Results: Together with reported chromosomal abnormalities, we found ABL gene exhibited not as BCR-ABL fusion gene in CML, but its amplication was prevalent, 67 patients (66.3%) had cytogenetic abnormalities with ABL amplication. And the patients with ABL gene amplication indicates no signicance with clinical features, adverse cytogenetics (C-MYC amplication, IGH rearrangement, RB1 deletion, P53 deletion and 1q21 amplication) and overall survival comparing to patients with normal ABL expression. Moreover, we revealed ABL amplication in MM cell lines (LP-1 and U266) by FISH, and ABL protein was easy to detect in MM cell lines and some tumor cells. According to CML cells, the cytotoxicity of STI571to MM cells was denitely limited. Conclusions: Our study rst discussed ABL gene amplication in MM cells, and we believe ABL gene would potentially be a useful target in the treatment of combination strategy for MM with ABL amplication in future.


Introduction
Multiple myeloma (MM) as one of the most common tumors of hematology, is characterized by malignant proliferation of plasma cells. Despite encouraging therapeutic advances, this disease remains an incurable disease due to complex genomic alterations, lower sensitivity to chemotherapy of MM cells in the bone marrow microenvironment and the emergence of drug resistance [1] Accumulating evidences have suggested that genotypic changes are found in 60% of patients at diagnosis by conventional chromosome analysis (CC) and in up to 90% of patients by uorescence in situ hybridization (FISH) analysis. Some of these genetic abnormalities have been identi ed: rearrangement of the 14q32 (IgH) locus\c-myc\cyclin D1\FGFR3\ cyclin D3, monoallelic deletions of chromosome 13, mutations of the Kras and N-ras genes, p53 monoallelic loss [2]. In addition, using DNA microarrays, a study compared the gene expression pro les of highly puri ed malignant plasma cells from nine patients with MM and eight myeloma cell lines to those of highly puri ed nonmalignant plasma cells (eight samples) obtained by in vitro differentiation of peripheral blood B cells. Overall there are two hundred and fty genes were signi cantly up-regulated and 159 down-regulated in malignant plasma samples compared to normal plasma samples. For some of these con rmed genes, ABL gene over-expressed in myeloma cells code for enzymes that could be a therapeutic target with speci c drugs [3].
ABL genes were rst identi ed in the guise of a tumor gene in the Abelson murine lymphosarcoma virus, and ABL-family proteins comprise one of the best conserved branches of the tyrosine kinases. The product of the virally transduced oncogene (v-ABL), was determined to be an altered form of cellular ABL (encoded by the c-ABL gene). ABL1 includes nuclear localization signals and a DNA binding domain through which it mediates DNA damage-repair functions, whereas ABL2 (also known as ABL related gene or Arg) has additional binding capacity for actin and for microtubules to enhance its cytoskeletal remodeling functions [4][5][6]. ABL genes are activated by chromosome translocations in various hematopoietic malignancies. Chronic myeloid leukemia (CML) is characterized in almost all cases by a t(9;22)(q34;q11) translocation. Importantly, transformation by ABL fusion proteins is inextricably tied to their tyrosine kinase activity, and targeted kinase inhibitor (Imatinib, also known as STI571or Gleevec) is therapeutically useful [7,8].Wild-type ABL is localized both in the nucleus and cytoplasm, and MM cells display high levels of nuclear ABL in response to ongoing DNA damage and genomic instability. However most of its nuclear tumor suppressor functions are compromised because of the disruption of the ABL-YAP1-p73 axis.Low YAP1 levels prevent nuclear ABL-induced apoptosis due to endogenous DNA damage,identifying a new synthetic-lethal strategy to selectively target cancer cells [9,10].
DNA FISH techniques have become increasingly popular among genome biologists, now widely accepted as the primary methodology for the validation of Hi-C results. Thanks to the direct observation of the three-dimentional (3D) genome architecture in a manner that is complementary to chromosome conformation capture methods such as Hi-C [11,12], as well as FISH probes produced, FISH is a versatile and expandable resource to study genome architecture, which can greatly facilitate the research and diagnostics of diseases.
Here, we rst used FISH method to do chromosomal pro ling and reported detailed ABL expression in MM bone marrow samples and MM cell lines. The performance of ABL gene ampli cation frequently appears even 3-fold or 4-fold in MM cells. Comparing to CML with BCR-ABL fusion gene, STI57 has poor anti-tumor effects in MM cells with ABL ampli cation through cell proliferation tests, western blot and FISH. Evidences have identi ed ABL is a selectively target of the synthetic-lethal strategy in MM.
Information on ABL regulatory mechanisms is being mined to provide new therapeutic strategies against hematopoietic malignancies not only BCR-ABL related types [13,14]. Therefore, even ABL gene has been described in tumors as the response to DNA damage, we believed its role in the combination therapies.

CCK8
Cells were seeded into 96-well plates and incubated with various drug concentrations in triplicates for 48 h. Cell proliferation was assayed using a Cell Counting Kit (CCK8) (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer's instructions. Each experiment was conducted in triplicate and repeated three times.

Western Blot
Cells were harvested, washed with PBS and lysed with lysis buffer (62.5 mM Tris-HCl, pH 6.8, 100 mM DTT, 2% SDS, 10% glycerol). Cell lysates were centrifuged at 20,000 g for 10 minutes at 4uC, and proteins in the supernatants were quanti ed. Protein extracts were equally loaded to 6% to 15% SDSpolyacrylamide gels, electrophoresed, and transferred to nitrocellulose membrane (Amersham Bioscience, Buckinghamshire, United Kingdom). After blocking with 5% nonfat milk in PBS for 2 hours at room temperature, the membranes were incubated with antibodies againstc-ABL was purchased from Santa Cruz Biotechnology (Santa Cruz, CA) overnight at 4℃, followed by HRP-linked secondary antibody for 1 hour at room temperature. The signals were detected by chemiluminescence phototope-HRP kit (Millipore) according to manufacturer's instructions, and α-tubulin/β-actin (Merck, Darmstadt, Germany) was probed as an internal control.

Statistical analysis
Depending on the distribution, the continuous data were presented as median (25th to 75th percentiles) or as mean ± SD. Categorical data were presented as counts or proportions. The differences between the groups were assessed with the χ 2 test or the Fisher's exact test for categorical data and the nonparametric Wilcoxon rank-sum test or a Student's t-test for continuous data. Univariate and multivariate logistic regression analyses were used to determine the relationship between ABL ampli cation and normal ABL expression groups. The analyses were performed using Empower (R) (www.empowerstats.com, X&Y Solutions, Inc. Boston MA) and R (http://www.R-project.org). And survival curves were constructed according to the Mantel-Cox method and compared using the log-rank test. A two-tailed value of P < 0.05 was considered to indicate statistical signi cance.

ABL ampli cation is frequent in MM patients
Between June 2009 and September 2018, 101 multiple myeloma patients (including 11 relapsed ones) with details were enrolled in the study, and we detected cytogenetic characteristic for each sample's bone marrow cells. The patients' baseline demographic and clinical features are summarized in Table 1. Among the MM patients, 67 patients (66.3%) had cytogenetic abnormalities with ABL ampli cation identi ed by FISH. Here, no signi cant differences were observed between the ABL ampli cation and normal ABL expression groups with regard to sex, age, M protein, Durie-Salmon (DS) stage, LDH, creatinine, albumin, β 2 -MG level, and karyotype (Table 1). In terms of cytogenetic features, we analyzed the incidences of C-MYC ampli cation, IGH rearrangement, P53 deletion, and 1q21 ampli cation in the ABL ampli cation group were higher than those in the normal ABL expression group, while the incidences of RB1/D13S319 deletion of in the ABL ampli cation group were lower than those in the normal ABL expression group (56.7% vs 64.8%). However, no signi cant differences in those cytogenetic abnormalities (shown in Table 1) were observed between the two groups (P 0.05). In contrast, signi cant differences in karyotype performance were found between the two groups. Hyperdiploidy indicating better prognosis [15] was more likely to be found in ABL ampli cation group, with the incidence was 32.8% (22/67), whereas the incidence in normal ABL expression group was 5.9% (2/34). The incidence of normal karyotype in ABL ampli cation group or ABL normal expression group were 65.7% (44/67), and 76.5% (26/34), respectively. However, the incidence of hypodiploid implying poor prognosis [16] was 17.6% (6/34) for normal ABL expression group and none in ABL ampli cation group. In addition, we found only one sample exhibited polyploidy feature, coexisting with ABL ampli cation, C-MYC ampli cation, IGH rearrangement, P53 deletion, and 1q21 ampli cation. We concluded ABL ampli cation was associated with genetic abnormalities, and myeloma cells were easy to nd ABL ampli cation in nucleus as the response of DNA injuries, particularly in cells withhyperdiploidy. Additionally, we further analyzed differences in overall survival (OS) between the two groups and 7 patients were lost to follow up (4 of ABL ampli cation group and 3 of normal ABL expression group). The results showed that the median survival of ABL ampli cation group and normal ABL expression group were 25 months and 34 months,but no signi cant difference was observed (P 0.05) (Figure 1).

Chromosomal characteristics of MM cell lines
To further assess the performance of FISH probes targeting small genomic loci in MM cell lines, we observed three human myeloma cell lines NCI-H929, U266 and LP-1 (they are characterized by hyperdiploidy karytype for 61-69 chromosomes and a variety of structural abnormalities) and CML cell line K562 with hyperdiploidy karytype and multi BCR-ABL fusion gene ampli cation. We detected C-MYC, RB1/D13S319, P53/1q21, IGH and BCR/ABL probes in NCI-H929, U266 and LP-1 cells. As shown in table 2, we found ABL ampli cation in LP-1 cells (three or four red signals of ABL gene signifying ABL gene ampli cation) and U266 cells (four red signals of ABL gene standing for of ABL gene ampli cation), while NCI-H929 plays normal ABL expression. According to Visualization of chromosomal territories by FISH chromosome-spotting probes, those high-risk genes were obtained based on Hi-C measurements. In NCI-H929 cells, C-MYC, IGH, D13S319/RB1 and 1q21 genes appeared triple ampli cation. In LP-1 cells, MYC ampli ed 6-fold, 1q21 ampli ed 8-10-fold and IGH rearranged. In U266 cells, C-MYC quadruple ampli ed, 1q21 ampli ed 6-fold, while variable region of IGH, P53 and D13S319/RB1 deleted. In Figure 2, C-MYC ampli cation, IGH rearrangement, P53 deletion, 1q21 ampli cation were con rmed in the metaphase or interphase FISH in these MM cell lines. In addition, RB1/D13S319 has differently chromosomes abnormalities, which ampli es in NCI-H929, normal in LP-1 and high ratio of de ciency in U266. Here, K562 cells were detected multi-BCR-ABL fusion gene ampli cation by FISH as the positive control.
3. ABL expression in MM cell lines ABL genes are found in all metazoans, including ABL fusion genes can transform human broblasts in cultures, and enhanced ABL signaling may contribute to epithelial cell malignancies, as well as to the invasive growth of breast cancer cells [17][18][19]. We then assessed ABL protein expression in both hematologic cells and solid tumors, and relatively easily found in most cells (Figure 3). The c-ABL protein expression could be identi ed in NCI-H929, LP-1, U266 and RPMI 8226 cell lines, while none of these cells exhibit BCR-ABL proteins. Even as described above, FISH was used to analyze ABL expression in nucleus of MM cell lines, and NCI-H929 does not show ABL ampli cation. There is not only nuclear c-ABL, but also cytoplasmic c-ABL, therefore, we know ABL protein level can be not consist with FISH performance.
3. The cytotoxity of ABL inhibitor STI571to MM cells We took advantage of ABL kinase inhibitor STI571 (Imatinib) cultured with MM cells, and found that the STI571 inhibitory concentration necessary to obtain a 50% inhibition in MM cell proliferation (IC 50 ) is rather high ( 10 μM ) (Figure 4) compared to the BCR-ABL fusion gene expressing CML cell line K562 (0.5 μM) (data not shown), which is consist with previous research [20]. And as shown in Figure 4, ABL protein expressions were dramatically blocked by the STI571 in MM cell lines. In addition, we used FISH to detect ABL gene performance in cell nucleus, we just found the number of genetic loci indicating BCR-ABL fusion genes reduced in high concentration of STI571 cultured with K562 cells for long time (Figure 4C), and rarely impact ABL ampli cation in MM cells (data not shown). STI571 appears targeting BCR-ABL mainly in cytoplasm because it works by binding close to the ATP binding site, hardly interferes ABL gene expression in cell nucleus [21]. The results implied that ABL kinase inhibitor can inhibit MM cells proliferation weakly comparing to CML cells, due to different mechanism of tumor cells development, and in the case of synergic effect on anti-myeloma, STI571 may work in some extent.

Discussion
MM is a genetically heterogeneous disease with a diverse clinical outcome and increasingly genetic abnormality will be explored in MM researches and diagnostics due to DNA technique development. Copy number alterations (CNAs), including whole chromosome and sub-chromosomal gains and losses, are common contributors of the pathogenesis and have demonstrated prognostic impact in MM [22]. In addition, diverse genomic landscapesingle nucleotide polymorphisme-array and next-generation sequencing (NGS) will extensively and deeply been pro led. At present, FISH a publically available resource enabling versatile DNA expression to study genome architecture, is a powerful method to study chromosomal organization in single cells. The novel method is reliable and can provide comprehensive pro ling of disease-related unbalanced genetic aberrations with a short turn-around time, such as gene deletion, ampli cation, inversion and fusion gene detection. On the basis of these features, FISH could represent a valuable addition to diagnostic methods currently used for the genetic characterization of MM, and provide the risk grades to indicate the prognosis of the disease, particular the patients with therapy including Bortezomib.
BCR-ABL fusion gene is involved in the Philadelphia chromosome in chronic myeloid leukemia, and rarely reported in myelomas. Even previous researches have con rmed over-expression of ABL gene in malignant comparing to normal plasma cells with the Mann-Whitney nonparametric statistical test [3]. Here, we have shown the gene ABL ampli cation was pervasive in MM patients and myeloma cell lines by FISH, and ABL protein expression also was easy to detect in MM cells and other tumor cells. ABL gene ampli cation was more common in MM cells with hyperdiploid karyotypes, which did not occur alone and was often accompanied by known high-risk genetic abnormalities, including C-MYC ampli cation, IGH rearrangement, P53 deletion, and 1q21 ampli cation. Consisting with previous research [20], our results suggested TKI did not work in ABL ampli cation MM cells as its role in BCR-ABL positive cells.
Although ABL kinase inhibitor STI571 revealed the weak cytotoxic effect to MM cells alone, it could be a therapeutic target with speci c drugs. This is the rst article on the discussion the role of ABL ampli cation in MM. Even we con rmed nucleus ABL expression in MM patients, it is regret that we did not further sort cells by CD138 magnetic beads, which can show better validation of chromosome loci abnormality. This will be explored by a further study based on CD138 expressions immunophenotyping enrichment to assess the sample differences. As ABL expression is prevalent in MM cells, a response to DNA damage, we found no signi cant clinical manifestation and survival differences between ABL ampli cation groups and ABL normal expression groups. And this result needs more samples to con rm. It is de cient we did not analyze the relationship of ABL gene ampli cation and detailed prognosis (including progression free survival) for MM patients due to the different chemotherapies and poor or incomplete compliance. And the following work will be improved. This is of particular interest since most tumor cells have a constitutive ABL activation, while TKI just plays apparently cytotoxic effect in few hematologic malignant tumors with BCR-ABL fusion gene, including CML, myelodysplastic syndrome (MDS)[23] and MM with BCR-ABL-positive [24]. However, combination of TKI and anti-IL-6 antibodiesinduced a marked and signi cant inhibition on myeloma cells proliferation at low concentrations [3]. In MM, as well as other hematologic and solid malignancies, genomic instability, centrosome ampli cation and aneuploidy have been associated with the overexpression of Aurora kinases, a family of serine/threonine kinases that play essential and distinct roles in mitosis [25]. Accordingly, combined inhibition of Aurora and ABL kinases resulted in substantial cell death and tumor regression in MM related to NF-κB-inducing kinase (NIK)-c-ABL-STAT3 signalingcentered feedback loop [13]. Conversely, Zhen Caiet al indicated activation of c-ABL Kinase could potentiate the anti-myeloma drug Lenalidomide by promoting DDA1 protein recruitment to the CRL4 ubiquitin ligase. Furthermore, Panobinostat (HDAC inhibiotr) and Dexamethasone can enhance Lenalidomide-induced substrate degradation and cytotoxicity by activating c-ABL, providing a mechanism underlying their combination with Lenalidomide to treat MM [14]. In the past decade, three-or even four-drug regimens have been increasingly popular recommended for myeloma patients, ABL kinase drugs can theoretically be applied. ABL includes nuclear localization signals and a DNA binding domain through which it mediates DNA damage-repair functions, whereas ABL in cytoplasm has additional binding capacity for actin and for microtubules to enhance its cytoskeletal remodeling functions. Several types of posttranslational modi cations control ABL catalytic activity, subcellular localization, and stability, with consequences for both cytoplasmic and nuclear ABL functions. Binding partners provide additional regulation of ABL catalytic activity, substrate speci city, and downstream signaling [4]. Therefore, information on ABL regulatory mechanisms is being further mined to provide new therapeutic strategies against hematopoietic malignancies with not only BCR-ABL fusion gene expression..

Conclusions
In conclusion, we used publically available and accepted technology FISH to study genome abnormalities in cells from MM patients'bone marrow, and rst identi ed the gene ABL ampli cation is frequent in MM. However, ABL ampli cation did not have the relationship with clinical syndrome and outcome. Furthermore, we detected ABL expression through FISH and western blot in vitro, and easy to nd ABL gene ampli cation and protein expression. Even the cell proliferation impacted by ABL kinase inhibitor STI571 is limited, we believe ABL gene would potentially be a useful target in the treatment of combination strategy for MM in future.  Tables   Table 1 Comparison     Copy number abnormalities were detected by FISH in MM cell lines. GLP IGH dual color breakpoint probe (located at 14q32), P53/1q21 probe (located at 17p13.1/1q21), D13S319/RB1 probe (located at 13q14), GLP C-MYC dual color breakpoint probe (located at 8q24), and GLP BCR-ABL dual color fusion probe (located at 22q11/9q34). A. NCI-H929: In interphase FISH, the picture displayed three fusion signals indicating IGH gene ampli cation, two green signals and three red signals signifying P53 gene normal and 1q21 amplifying. In metaphase FISH, the picture displayed three green and three red signals indicating D13S319 RB1 gene ampli cation. B. LP1: In interphase FISH, the picture displayed two fusion, six green and two red signals indicating IGH gene rearrangement and ampli cation, three green signals and ten red signals signifying P53 gene and 1q21 amplifying. In metaphase FISH, the picture displayed two green and two red signals indicating D13S319 RB1 gene normal, but it can show chromosome 13 transformed into derivative chromosome 13. And the picture displayed three green and three red signals indicating BCR ABL gene ampli cation. C. U266: In interphase FISH, the picture displayed in diploid cells, one fusion and one red signals indicating IGH gene rearrangement and IGH variant region deletion. In tetraploid cells, two fusion and two red signals, indicated IGH gene rearrangement and IGH variant region deletion. And the picture displayed four green and four red signals indicating BCR ABL gene ampli cation, four fusion signals indicating MYC gene ampli cation. In metaphase FISH, the picture showed two green signals and six red signals signifying P53 gene deletion and 1q21 amplifying, two green and two red signals indicating D13S319 RB1 gene normal. D. K562: In metaphase FISH, the picture displayed two idic (Ph), another one fusion signal three red ABL and two BCR genes. And in interphase FISH, the picture displayed multiple fusion genes and three red ABL and two BCR genes.   with STI571 for 48 h. C. The gene changes impacted by STI571 measured by FISH analysis with GLP BCR-ABL dual color fusion probe (located at 22q11/9q34).In interphase FISH, the picture displayed multiple fusion genes, three red ABL and two BCR genes. BCR-ABL fusion genes exhibited no change and slightly reduced when add 20μM and 40 μM STI571 for 60h, respectively.