scholarly journals BCR-ABL1 p190 in CML: A Minor Breakpoint with a Major Impact

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 190-190
Author(s):  
Shady Adnan Awad ◽  
Helena Hohtari ◽  
Komal Kumar Javarappa ◽  
Tania Brandstoetter ◽  
Daehong Kim ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Exome Sequencing ◽  
Rna Sequencing ◽  
Cell Lines ◽  
Research Funding ◽  
Drug Sensitivity ◽  
Genetic Alterations ◽  
Resistance Testing ◽  
Sequencing Analysis ◽  
Distinct Features

Introduction: The oncoprotein Bcr-Abl has two major isoforms, depending on the breakpoint in BCR gene, p190 and p210. While p210 is the hallmark of chronic myeloid leukemia (CML), p190 occurs in the majority of Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL) patients. p190 occurs as a sole transcript in 1-2% CML patients, associated with distinct features like monocytosis and frequent additional cytogenetic abnormalities (ACA) at diagnosis. It also confers a risk of treatment failure and progression in chronic phase (CP) CML patients. However, the underlying mechanisms are largely unknown. Here we explore the characteristics of p190 and p210 in CML and ALL patients using next generation sequencing, phospho-flowcytometry and high throughput drug testing. Patients and methods: Peripheral blood mononuclear cells (PMNC) were collected at diagnosis from four CP-CML patients harboring p190 isoform from Helsinki University Hospital. Genetic alterations were identified by whole exome sequencing. RNA sequencing was employed to analyze transcriptional profiles of p190 CML (n=3) in contrast to p210 CML patients (n=4). A thorough transcriptional, phosphorylation and drug sensitivity profiling were applied to five p190- and three p210-expressing Ph+ALL patients. Expression alterations were further characterized in two cell line models mimicking BCR-ABL positive leukemia (Ba/F3 and HPCLSK). Phosphorylation profiles were analyzed by flowcytometry and phospho-array (Tyrosine Phosphorylation ProArray, Full Moon Biosystems). For drug sensitivity and resistance testing (DSRT), a custom plate set comprising 75 approved and investigational oncology drugs was used for patient samples and more extensive 528-drugs plates were used for the cell lines. Results: CML patients with p190 had a median age of 72.5 years at the diagnosis (range: 50-80) and all received imatinib as a frontline treatment. Only one patient achieved a fluctuating major molecular response (MMR) by 12 months while the rest of the patients showed primary resistance to treatment and were shifted to a 2nd line TKI, nilotinib (n=2) or proceeded to HSCT (n=1). By exome sequencing we identified 26 variants in p190-CML samples (median per patient=7, range: 2-10), including variants in ASXL1, DNMT3A and KDM4D genes. RNA-sequencing analysis identified 19 and 97 dysregulated genes (Q <0.05) between p190- and p210 in CML and Ph+ ALL cells respectively. In CML, enrichment analysis revealed upregulation of TNF, interferon (IFN), IL1-R and Toll-like receptor (TLR) signaling, TP53-related, cell cycle and apoptosis pathways. Among Ph+ ALL samples, many CML-related genes were upregulated in samples encompassing p210 while IFN-, TP53- and cell cycle-related molecules were upregulated in p190 samples. p190 samples exhibited hyper-phosphorylation of Src kinase compared to p210 samples. DSRT results also revealed increased sensitivity of primary Ph+ ALL-p190 cells to Src-inhibitors (dasatinib and saracatinib), glucocorticoids and MDM2 inhibitors/TP53 activators (SAR405838 and idasanutlin). Regarding cell lines, Ba/F3-p190 showed the upregulation of interferon signaling pathways compared to p210. Src was also hyperphosphorylated in both Ba/F3 and HPCLSK p190 models. In addition to glucocorticoids and Src-inhibitors, compounds blocking the activity of the inhibitors of apoptosis protein (IAP) family were highly effective at reducing the viability of p190 compared to p210 cells in both cell lines. Conclusions: In CML, p190 isoform of BCR-ABL1 is associated with distinct features and should be considered as a high-risk group. Combining clinical, genomic, phosphorylation and drug sensitivity data, we demonstrated that p190 activates specific cancer pathways, notably Src signaling and interferon pathways. Data also suggests that CML patients with p190 could benefit from broad spectrum TKI with Src inhibiting activity or combination of TKI with MDM2- or IAP-inhibitors. Disclosures Heckman: Orion Pharma: Research Funding; Celgene: Research Funding; Novartis: Research Funding; Oncopeptides: Research Funding. Porkka:Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding. Mustjoki:Novartis: Research Funding; Pfizer: Research Funding; BMS: Honoraria, Research Funding.

Exome Sequencing of Aggressive Natural Killer Cell Leukemia and Drug Profiling Highlight Candidate Driver Pathways in Malignant Natural Killer Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 700-700
Author(s):  
Olli Dufva ◽  
Tiina Kelkka ◽  
Shady Awad ◽  
Nodoka Sekiguchi ◽  
Heikki Kuusanmäki ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Exome Sequencing ◽  
Cell Lines ◽  
Hierarchical Clustering ◽  
Research Funding ◽  
Drug Sensitivity ◽  
Somatic Mutations ◽  
Nk Cell ◽  
Negative Regulator

Abstract Background Natural killer (NK) cell malignancies are rare lymphoid neoplasms characterized by aggressive clinical behavior and poor treatment outcomes. Clinically they are classified as extranodal NK/T-cell lymphoma, nasal type (NKTCL) and aggressive NK cell leukemia (ANKL). Both subtypes are almost invariably associated with Epstein-Barr virus (EBV). Recently, genomic studies in NKTCL have identified recurrent somatic mutations in JAK-STAT pathway molecules STAT3 and STAT5b as well as in the RNA helicase gene DDX3X in addition to previously detected chromosomal aberrations. Here, we identified somatic mutations in 4 cases of ANKL in order to understand whether these entities share common alterations at the molecular level. To further establish common patterns of deregulated oncogenic signaling pathways operating in malignant NK cells, we performed drug sensitivity profiling using NK cell lines representing ANKL, NKTCL and other malignant NK cell proliferations. We aimed to identify sensitivities to agents that selectively target components of pathways required for survival of malignant NK cells in an unbiased manner. Methods Exome sequencing was performed on peripheral blood or bone marrow of ANKL patients using the NK cell negative fraction or other healthy tissue as control. Profiling of drug responses was performed with a high-throughput drug sensitivity and resistance testing (DSRT) platform comprising 461 approved and investigational oncology drugs. The NK cell lines KAI3, KHYG-1, NKL, NK-YS, NK-92, SNK-6 and YT and IL-2-stimulated and resting NK cells from healthy donors were used as sample material. All drugs were tested on a 384-well format in 5 different concentrations over a 10,000-fold concentration range for 72 h and cell viability was measured. A Drug Sensitivity Score (DSS) was calculated for each drug using normalized dose response curve values. Results The ANKL patients displayed mutations in genes reported as recurrently mutated in NKTCL, such as FAS, TP53, NRAS, STAT3 and DDX3X. Additionally, novel alterations in genes previously implicated in the pathogenesis of NKTCL were detected. These included an inactivating mutation in INPP5D (SHIP), a negative regulator of the PI3K/mTOR pathway and a missense mutation in PTPRK, a negative regulator of STAT3 activation. Interestingly, the total number of nonsilent somatic mutations in 3 out of 4 ANKL patients (97, 82 and 45) was remarkably high compared to other hematological malignancies analyzed in our variant calling pipeline. Analysis of drug sensitivities in NK cell lines showed a close correlation between all cell lines and a markedly higher correlation with those of IL-2 stimulated than resting healthy NK cells, suggesting that malignant NK cells may share a common drug response pattern. Furthermore, in an unsupervised hierarchical clustering the NK cell lines formed a distinct group from other leukemia cell lines tested (Fig. A). Among pathway-selective compounds (namely, kinase inhibitors and rapalogs), the drugs most selective for malignant NK cells fell into two major categories: PI3K/mTOR inhibitors (e.g. temsirolimus, buparlisib) and inhibitors of aurora and polo-like kinases such as rigosertib and GSK-461364 (Fig. B). JAK inhibitors (e.g. ruxolitinib, gandotinib) and CDK inhibitors (e.g. dinaciclib) showed strong efficacy in both malignant NK cells and IL-2 activated healthy NK cells. Conclusions Our exome sequencing results suggest that candidate driver alterations affecting similar signaling pathways underlie the pathogenesis of ANKL as has been reported in NKTCL. Drug sensitivity profiling highlights the PI3K/mTOR pathway as a potential major driver of malignant NK cell proliferation, whereas JAK-STAT signaling appears to be essential in both healthy and malignant NK cells. Components of these pathways harbored mutations in our small cohort of ANKL patients and have been shown to be deregulated by mutations or other mechanisms in previous studies, underlining their importance as putative drivers. The systematic large-scale characterization of drug responses also identified these pathways as potential targets for novel therapy strategies in NK cell malignancies. Figure 1. (A) Unsupervised hierarchical clustering based on drug sensitivity scores (DSS) of NK, AML, CML and T-ALL cell lines. (B) Scatter plot comparing DSS of malignant NK cell lines (average) and healthy IL-2 stimulated NK cells. Figure 1. (A) Unsupervised hierarchical clustering based on drug sensitivity scores (DSS) of NK, AML, CML and T-ALL cell lines. (B) Scatter plot comparing DSS of malignant NK cell lines (average) and healthy IL-2 stimulated NK cells. Disclosures Mustjoki: Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding.

scholarly journals RUNX1 Mutations Identify an Entity of Blast Phase Chronic Myeloid Leukemia (BP-CML) Patients with Distinct Phenotype, Transcriptional Profile and Drug Vulnerabilities

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4257-4257 ◽  
Author(s):  
Shady Awad ◽  
Matti Kankainen ◽  
Olli Dufva ◽  
Caroline A Heckman ◽  
Kimmo Porkka ◽  
...  
Keyword(s):  
Cell Line ◽  
Rna Sequencing ◽  
Research Funding ◽  
Mtor Inhibitors ◽  
Stem Cell Differentiation ◽  
Genetic Alterations ◽  
Group Signature ◽  
Resistance Testing ◽  
Control Group ◽  
Knock Out

Abstract Introduction: Genetic alterations of RUNX1 gene (mutations and fusion genes) are common in hematological malignancies including AML, ALL, MDS, and MPN. Mutations of RUNX1 occur in 10% of newly diagnosed AML patients and associate with inferior prognosis. Similarly, point mutations in RUNX1 are frequent in BP-CML, but little is known about their role in BP patients. Methods: Bone marrow samples from four BP patients with RUNX1 mutations were collected from Helsinki University Hospital, and samples from four other BP patients without RUNX1 mutations were used as controls. Samples were analyzed by exome and RNA sequencing. For drug sensitivity and resistance testing (DSRT), a high-throughput platform comprising 195 approved and investigational oncology drugs was used. To further study the role of RUNX1 mutations in BP-CML, Baf3/BCR-ABL1+ RUNX1-/- knock-out and RUNX1-/mut heterozygous deletion mutant models were created using CRISPR-CAS9 technology. The effects of the mutations in the cell line models were characterized by flow cytometry, RNA sequencing, and DSRT analyses. Results: The median age of RUNX1-mutated BP patients was 47 years (range 36-56 years) and included 3 patients with myeloid-BP and 1 with lymphoid-BP phenotype. RUNX1-mutated patients tended to have higher mutational load and co-occurrence with mutations to the BCOR, EZH2 and PHF6 genes in agreement with the mutational profile of RUNX1-mutated AML. Signature analysis of RUNX1-mutated BP patients showed dominance of signature 1 (age-related), signature 6, and 15 (DNA mismatch repair) correlating with the control group signature profiles. In addition, the RUNX1 mutated group showed specific enrichment of signature 9, related to somatic hypermutation and AID/RAG axis activity, that was not observed in the control group. RNA sequencing revealed that 388 genes were significantly differentially expressed between RUNX1-mutated and control BP patients (139 upregulated, 249 downregulated). Interestingly, the antigen processing and presentation pathway was among the top upregulated pathways in RUNX1 mutated patients with overexpression of CIITA and HLA-DR genes. Stem cell differentiation, complement, and hemostasis pathways were enriched in the downregulated gene set. Lymphoid-specific transcription factors (DNTT, PAX5 and VPERB1) in addition to CD19 and CD25 were significantly upregulated in the RUNX1-mutated group. DSRT data showed greater sensitivity to glucocorticoids, MEK-, mTOR- and VEGFR-inhibitors in RUNX1-mutated patients compared to the control group. Data from RUNX1-/- and RUNX1-/mut cell line models confirmed the induced expression of CD19 surface marker with RUNX1 mutation. DSRT results showed increased activity of MEK inhibitor and mTOR inhibitors in RUNX1-/- knock-out and RUNX1-/mut cell lines. Conclusions: RUNX1 mutations in BP-CML patients associate with distinct phenotypic and transcriptional features. DSRT results together with transcriptional data support the potential benefits of glucocorticoids and MEK and mTOR inhibitors as well as immunotherapy targeting CD19 and CD25 in this group of BP-CML patients. Disclosures Kankainen: Medix Biochemica: Consultancy. Heckman:Celgene: Research Funding; Orion Pharma: Research Funding; Novartis: Research Funding. Porkka:Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Mustjoki:Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Ariad: Research Funding; Celgene: Honoraria.

High-Throughput Drug Sensitivity and Resistance Testing (DSRT) Platform Reveals Novel Candidate Drugs For Advanced Phase BCR-ABL1-Positive Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2719-2719
Author(s):  
Paavo Pietarinen ◽  
Muntasir Mamun Majumder ◽  
Disha Malani ◽  
Astrid Murumägi ◽  
Tea Pemovska ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cell Lines ◽  
High Throughput ◽  
Research Funding ◽  
Drug Sensitivity ◽  
Protein Translation ◽  
Resistance Testing ◽  
Antibody Array ◽  
Secondary Resistance ◽  
Advanced Phase

Abstract Background Advanced BCR-ABL1-positive leukemias (chronic myeloid leukemia in blast crisis and Ph+ALL) remain a therapy challenge despite advances in tyrosine kinase inhibitor (TKI) therapy. Emergence of primary and secondary resistance due to gatekeeper and compound mutations within the BCR-ABL1 kinase domain is common even with the novel 2nd and 3rd generation TKIs (dasatinib, nilotinib, ponatinib). We set out to identify novel candidate drugs for advanced BCR-ABL1-positive leukemias by using an unbiased high-throughput drug testing platform and utilizing both primary patient cells and cell lines. Methods As a study material we used 3 CML cell lines representing different types of CML blast phases. In addition to commonly used K562 cells, EM-2 and MOLM-1 cell lines were tested. AML cell lines (AML-193, AP-1060, HL60ATCC, HL60TB, Kasumi-1, KG-1, ME-1, MOLM-13, MONO-MAC-6, MUTZ-2, MV4-11, NOMO-1, SH-2, SHI-1, SIG-M5, SKM-1, THP-1) were used as cell line controls. To verify the results obtained from cell lines, primary bone marrow (BM) cells were derived from 2 TKI-resistant CML BC patients. Patient 1 had developed resistance to imatinib and dasatinib due to a T315I mutation, whereas patient 2 was resistant to nilotinib, dasatinib and ponatinib due to a V299L and a compound mutation. BM cells from 4 healthy individuals were used as controls. The functional profiling of drug responses was performed with a high-throughput drug sensitivity and resistance testing (DSRT) platform comprising of 306 anti-cancer agents (FDA/EMA approved, investigational and experimental compounds). Cells were dispensed to pre-drugged 386-well plates of 5 different concentrations and incubated in a humidified incubator with 5% CO2 at 37 °C for 72 hours. Cell viability was measured by using a luminescent cell viability assay (CellTiter-Glo). From plate reads a Drug Sensitivity Score (DSS) was calculated for each drug as a measure of cytotoxicity. In addition to DSRT, Human Phospho-Kinase Array Kit (R&D systems) was used to analyze the phosphokinase profile in patient samples. Results Based on initial comparisons between CML and AML cells lines, nonspecific cytotoxic drugs, which showed high activity in all cell lines, were omitted from further analysis. The DSS scores from different CML cells lines correlated relatively closely (EM-2 vs. K-562, r=0.89; EM-2 vs. MOLM-1, r=0.82; K-562 vs. MOLM-1, r=0.78; p<0.0001 for all correlations). We next ranked the DSRT data according to the DSS values with most sensitive drugs showing the highest DSS scores. The primary cells from CML BC were further normalized against the median values from healthy controls, resulting in leukemia-specific sensitivity scores (sDSS). Ranked results from the DSRT analysis are shown in the Table. As expected, the cell lines were sensitive to TKIs, with the exception of the MOLM-1, which showed only modest sensitivity. The clinically TKI-resistant patient samples were also TKI-resistant ex vivo, further validating the DSRT assay data. Drugs which showed efficacy in both the cell lines and the TKI-resistant patients included HSP90 inhibitors (NVP-AUY922, BIIB021), a NAMPT inhibitor daporinad and the protein translation inhibitor omacetaxine (homoharringtonine). Phosphokinase antibody array results from the patient samples showed increased expression of the HSP27 protein as a putative biomarker for HSP90 inhibitor response. Conclusions DSRT is a powerful assay for identifying novel candidate molecules for refractory BCR-ABL1-positive leukemias. Our results indicate that HSP90 and NAMPT inhibitors in particular warrant further clinical evaluation both by analyzing a larger set of primary patient samples and by performing proof-of-concept clinical studies. The results also pave way for designing rational combination therapy strategies. Disclosures: Mustjoki: Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau. Porkka:BMS: Consultancy, Research Funding, Speakers Bureau; Novartis: Consultancy, Research Funding, Speakers Bureau.

AML Specific Targeted Drugs Identified By Drug Sensitivity and Resistance Testing: Comparison of Ex Vivo Patient Cells with in Vitro Cell Lines

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2163-2163
Author(s):  
Disha Malani ◽  
Astrid Murumägi ◽  
Bhagwan Yadav ◽  
Tea Pemovska ◽  
John Patrick Mpindi ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Drug Sensitivity ◽  
Ex Vivo ◽  
Cancer Cell Lines ◽  
Resistance Testing ◽  
Targeted Drugs ◽  
Drug Responses

Abstract Introduction Many drug discovery efforts and pharmacogenomic studies are based on testing established cancer cell lines for their sensitivity to a given drug or a panel of drugs. This approach has been criticized due to high selectivity and fast proliferation rate of cancer cell lines. To explore new therapeutic avenues for acute myeloid leukemia (AML) and to compare experimental model systems, we applied high-throughput Drug Sensitivity and Resistance Testing (DSRT) platform with 305 approved and investigational drugs for 28 established AML cell lines and compared their drug responses with our earlier study of 28 ex vivo AML patient samples (Pemovska et al., 2013). We then correlated drug sensitivities with genomic and molecular profiles of the samples. Methods DSRT was carried out with 305 clinical, emerging and experimental drugs and small molecule chemical inhibitors. The drugs were tested at five different concentrations over a 10,000-fold concentration range. Cell viability was measured after 72 hours using Cell Titre Glow assay. IC50 values were calculated with Dotmatics software and drug sensitivity scores (DSS, a modified area under the curve metric) were derived for each drug (Yadav et al., 2014). Nimblegen's SeqCap EZ Designs Comprehensive Cancer Design kit was used to identify mutations from 578 oncogenes in cell lines. Results The 28 established AML cell lines were in general more sensitive to the drugs as compared to the 28 ex vivo patient samples, with some important exceptions. Sensitivity towards many targeted drugs was observed in both AML cell lines and in patient samples. These included inhibitors of MEK (e.g. trametinib in 56% of cell lines and 36% of ex vivo samples), mTOR (e.g. temsirolimus in 42% and 32%) and FLT3 (quizartinib in 28% and 18%). Overall, drug responses between cell lines and ex vivo patient cells in AML showed an overall correlation coefficient of r=0.81. BCL2 inhibitors (venetoclax and navitoclax) showed more sensitivity in ex vivo patient cells than in AML cancer cell lines, whereas responses to anti-mitotic agents (docetaxel, camptothecin, vincristine) showed stronger responses in cell lines (Figure). Only 7% of AML cell lines exhibited responses to a broad-spectrum tyrosine kinase inhibitor dasatinib, in contrast to 36% patient samples. AML cell lines that carried FLT3 mutations showed high sensitivity to FLT3 inhibitors. Similarly, cell lines harbouring mutations in RAS or RAF were strongly sensitive to MEK inhibitors. MEK and FLT3 inhibitor responses were mutually exclusive, indicating alternative pathway dependencies in cell lines. However, these pharmacogenomics correlations were not as clearly seen in the clinical samples. Summary These data revealed a few important differences as well as many similarities between established AML cell lines and primary AML patient samples in terms of their response to a panel of cancer drugs. The hope is that patient-derived primary cells in ex vivo testing predict clinical response better as compared to the established cancer cell lines, which indeed seem to overestimate the likelihood of responses to many drugs. On the other hand, cancer cell line studies may also underestimate the potential of dasatinib and BCL2 inhibitors as emerging AML therapeutics. References 1. Pemovska T, Kontro M, Yadav B, Edgren H, Eldfors S, Szwajda A, et al. Individualized systems medicine strategy to tailor treatments for patients with chemorefractory acute myeloid leukemia. Cancer Discovery. 2013 Dec;3(12):1416-29 2. Yadav B, Pemovska T, Szwajda A, Kulesskiy E, Kontro M, Karjalainen R, et al. Quantitative scoring of differential drug sensitivity for individually optimized anticancer therapies. Scientific reports. 2014;4:5193. Figure: Correlation of average drug responses (n=305) between 28 AML cell lines and 28 AML ex vivo patient samples Figure:. Correlation of average drug responses (n=305) between 28 AML cell lines and 28 AML ex vivo patient samples Disclosures Heckman: Celgene: Research Funding. Porkka:BMS: Honoraria; BMS: Research Funding; Novartis: Honoraria; Novartis: Research Funding; Pfizer: Research Funding. Kallioniemi:Medisapiens: Consultancy, Membership on an entity's Board of Directors or advisory committees.

scholarly journals Exome Sequencing of Relapsed Multiple Myeloma Combined with Pooled CRISPR/Cas9 Screens Identifies Gene Mutations Associated with Drug-Specific Resistance

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 809-809
Author(s):  
Stephan Bohl ◽  
Laura K. Schmalbrock ◽  
Imke Bauhuf ◽  
Anna Dolnik ◽  
Tamara J. Blätte ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Exome Sequencing ◽  
Cell Lines ◽  
Research Funding ◽  
Specific Resistance ◽  
Gene Mutations ◽  
Genetic Alterations ◽  
Recurrent Mutations ◽  
Pre Treatment ◽  
Increased Susceptibility

Lenalidomide, bortezomib, melphalan and dexamethasone are standard drugs for the treatment of multiple myeloma (MM). Although many patients initially respond to treatment regimens including these drugs, the majority ultimately relapses due to the development of resistance of the MM cells, what may result from acquired genetic alterations. Here we performed fluorescence in situ hybridization (FISH) and whole exome sequencing (WES) on 16 paired pre-treatment/ progression MM samples followed by functional validation through CRISPR/Cas9-based screens to identify gene mutations that are associated with resistance. Treatment between two samples consisted of lenalidomide (n=16), bortezomib (n=14), dexamethasone (n=16) and melphalan (n=9). Cytogenetic analyses by FISH revealed that the majority of translocations (7 of 10) and chromosomal gains and deletions (22 of 28) were concordant between pre-treatment and relapse samples. In contrast, gene mutations assessed by WES were highly variable: of the total of 794 identified mutations 6% (n=46) were present only at diagnosis, 59% (n=474) at both time points and 35% (n=274) specifically at relapse with an increase of the median number of mutations from 29 (range 9-103) in pre-treatment to 47 (range 13-110) in progression samples (figure 1A). Recurrent mutations detected pretreatment were in general stable at progression: NRAS (3/3), KRAS (4/4), IGLL5 (3/3) and DIS3 (2/3). Only very few of the newly acquired gene mutations at progression were recurrent: TP53 (n=4), DNAH5 (n=4) and WSCD2 (n=3) while the remaining were non-recurrent. In order to investigate the functional impact of relapse-specific gene mutations on drug resistance we performed pooled CRISPR-Cas9-based knockout screens (figure 1B). We included 160 gene mutations that fulfilled the following criteria: 1) a variant allele fraction (VAF) of &gt;20% at the time of progression, 2) found exclusively in progression samples or had a more than 2-fold increase in VAF at progression as compared to pre-treatment, 3) predicted to be loss-of-function. In addition, we included genes found recurrently mutated in relapsed MM in previously published studies. Resistance screens were performed in three different MM cell lines (MM1S, NCIH-929, KMS27) with 4 sgRNA per gene in the presence of lenalidomide, dexamethasone, melphalan, bortezomib or DMSO as a control. None of the sgRNAs included in the screen conferred resistance to all four drugs. In contrast, we identified several genes whose inactivation caused resistance to a specific drug. For lenalidomide, the top hits were members of the CRBN-CRL4 E3 ubiquitin ligase, the primary target of IMiDs, including CRBN, CUL4B and DDB1. CRISPR-mediated inactivation of these genes was specifically associated with lenalidomide resistance since sensitivity towards other drugs was not affected. In addition, we found sgRNAs targeting SYT5, a membrane protein involved in Ca2+-dependent exocytosis, to be enriched in lenalidomide-treated cells that so far has not been related to lenalidomide resistance. SgRNAs targeting TP53 were also weakly enriched after lenalidomide treatment in two of the three cell lines but conferred a high level of resistance to melphalan in all three cell lines (figure 1C). Consistently, three of the four TP53 mutations identified by WES were detected in samples obtained after cytotoxic chemotherapy and one after 3 years of treatment with lenalidomide/dexamethasone. Our screens also revealed an increased susceptibility to melphalan by inactivation of ATM, FANCA, BIRC3, and BRCC3, all involved in DNA damage repair. The top sgRNAs causing resistance to dexamethasone were directed against ANKMY2 and BIRC3 in two cell lines (MM1S and NCI-H929). For bortezomib, inactivation of only one gene, TMC2, encoding a transmembrane protein was associated with resistance in two cell lines whereas BIRC3 inactivation provided increased susceptibility to bortezomib. In conclusion, by combination of comprehensive genetic analyses of tumor samples before and after treatment with functional genetic screens we found mutations that are causally linked with drug-specific resistance and sensitivity. These results may help to personalize therapy in patients with multiple myeloma. Figure 1 Disclosures Bohl: Pfizer: Honoraria. Döhner:Celgene, Novartis, Sunesis: Honoraria, Research Funding; AbbVie, Agios, Amgen, Astellas, Astex, Celator, Janssen, Jazz, Seattle Genetics: Consultancy, Honoraria; AROG, Bristol Myers Squibb, Pfizer: Research Funding. Bullinger:Astellas: Honoraria; Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Daiichi Sankyo: Honoraria; Gilead: Honoraria; Hexal: Honoraria; Janssen: Honoraria; Jazz Pharmaceuticals: Honoraria; Menarini: Honoraria; Novartis: Honoraria; Pfizer: Honoraria; Sanofi: Honoraria; Seattle Genetics: Honoraria; Bayer: Other: Financing of scientific research; Abbvie: Honoraria; Amgen: Honoraria. Krönke:Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Validation of the Function of 14-3-3 ζ in Multiple Myeloma (MM)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1369-1369
Author(s):  
Yanyan Gu ◽  
Jonathan L. Kaufman ◽  
Lawrence H. Boise ◽  
Sagar Lonial
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Signaling Pathways ◽  
Cell Lines ◽  
Research Funding ◽  
Drug Sensitivity ◽  
Stable Cell Lines ◽  
Induced Apoptosis ◽  
The Impact

Abstract Abstract 1369 The 14-3-3 protein family includes seven members, β, γ, ε, η, σ, τ and ζ. With over 200 binding partners, 14-3-3 proteins act as integrators of diverse cell signaling pathways and participate in metabolism, cell cycle regulation, survival and apoptosis. 14-3-3ζ has been implicated in many cancers such as hepatocellular carcinoma, gastric cancer, breast cancer, lung carcinoma and lymphoma. However, the role of 14-3-3ζ in MM has not been extensively explored. Preliminary data from an affymatrix GEP profile of normal plasma cells (NPC), MGUS, Smoldering myeloma (SM) or multiple myeloma (MM) demonstrates statistically increased expression of 14-3-3 ζ in the transition between MGUS and SM. Among patients with newly diagnosed symptomatic MM, 14-3-3 ζ expression appears to be higher in the higher risk genetic subsets. These data suggest 14-3-3ζ plays a prominent role in the biology of MM especially among high risk myeloma patients. In order to identify the impact of 14-3-3 ζ signaling on MM proliferation and survival, we developed 14-3-3ζ silenced and over expressing stable cell lines to interrogate the biological role of 14-3-3ζ in MM. Using a library of human MM cell lines, we found that 14-3-3ζ is universally expressed in all MM cell lines examined. Knockdown of 14-3-3ζ significantly inhibits cell growth and proliferation in LP1 and U266 cells, which is partly related to G1 cell cycle arrest. Relevant signaling proteins such as Mcl-1, Bcl2, phospho-Akt and CDK6 decrease after silencing 14-3-3ζ. Furthermore, we performed gene expression profiling of LP1 scrambled and knockdown stable cell lines in order to identify key changes in gene regulation that may be mediated via 14-3-3ζ. The GEP data suggests that 14-3-3ζ is responsible for but not limited to several important signaling pathways, such as glycolysis/gluconeogenesis, p53 Signaling, NRF2-mediated oxidative stress response and death receptor signaling. In addition, we evaluated the effect of 14-3-3ζ expression on the drug sensitivity to commonly used chemotherapeutic compounds in MM treatment, such as bortezomib, etoposide, dexamethasone, melphalan, lenalidomide, doxorubicin and romidepsin. Knockdown 14-3-3ζ sensitizes cells to romidepsin- induced apoptosis, as demonstrated by Annexin V staining and western blot assay for caspase cleavage. However, bortezomib- induced apoptosis is significantly inhibited when 14-3-3ζ is silenced. Bortezomib (5nM)-induced apoptosis decreased from 37% in LP1 cells expressing shRNA with scrambled sequence to 14% in LP1 cells where 14-3-3 ζ is silenced. Moreover, 14-3-3ζ knockdown effectively inhibits bortezomib induced NOXA upregulation, suggesting a possible new molecular mechanism for the effects of 14-3-3ζ in bortezomib mediated apoptosis. Taken together, our work reveals the important biological function of 14-3-3ζ in MM growth, survival and proliferation; the data also provides valuable information for the development of new therapeutic strategies facilitating drug sensitivity and overcoming drug resistance. Disclosures: Kaufman: Millenium: Consultancy; Onyx Pharmaceuticals: Consultancy; Novartis: Consultancy; Keryx: Consultancy; Merck: Research Funding; Celgene: Research Funding. Lonial:Onyx: Consultancy; Bristol-Myers Squibb: Consultancy; Novartis: Consultancy; Celgene: Consultancy; Millennium Pharmaceuticals, Inc.: Consultancy; Merck: Consultancy.

scholarly journals A chemical screen in diverse breast cancer cell lines reveals genetic enhancers and suppressors of sensitivity to PI3K isoform-selective inhibition

2008 ◽  
Vol 415 (1) ◽  
pp. 97-110 ◽  
Author(s):  
Neil E. Torbett ◽  
Antonio Luna-Moran ◽  
Zachary A. Knight ◽  
Andrew Houk ◽  
Mark Moasser ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Cycle ◽  
Cell Lines ◽  
Cancer Cell ◽  
Breast Cancer Cell ◽  
Cancer Therapeutics ◽  
Cancer Cell Lines ◽  
Genetic Alterations ◽  
Breast Cancer Cell Lines ◽  
Selective Inhibitors

The PI3K (phosphoinositide 3-kinase) pathway regulates cell proliferation, survival and migration and is consequently of great interest for targeted cancer therapy. Using a panel of small-molecule PI3K isoform-selective inhibitors in a diverse set of breast cancer cell lines, we have demonstrated that the biochemical and biological responses were highly variable and dependent on the genetic alterations present. p110α inhibitors were generally effective in inhibiting the phosphorylation of PKB (protein kinase B)/Akt and S6, two downstream components of PI3K signalling, in most cell lines examined. In contrast, p110β-selective inhibitors only reduced PKB/Akt phosphorylation in PTEN (phosphatase and tensin homologue deleted on chromosome 10) mutant cell lines, and was associated with a lesser decrease in S6 phosphorylation. PI3K inhibitors reduced cell viability by causing cell-cycle arrest in the G1 phase, with multi-targeted inhibitors causing the most potent effects. Cells expressing mutant Ras were resistant to the cell-cycle effects of PI3K inhibition, which could be reversed using inhibitors of Ras signalling pathways. Taken together, our data indicate that these compounds, alone or in suitable combinations, may be useful as breast cancer therapeutics, when used in appropriate genetic contexts.

scholarly journals Preclinical Activity of the MPS1 Inhibitor S81694 in Acute Lymphoblastic Leukemia (ALL)

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2631-2631
Author(s):  
Anna Kaci ◽  
Emilie Adiceam ◽  
Melanie Dupont ◽  
Marine Garrido ◽  
Jeannig Berrou ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Solid Tumors ◽  
Cell Lines ◽  
Small Molecule ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
Jurkat Cells ◽  
Cell Cycle Distribution ◽  
Monopolar Spindle

Introduction: The dual-specificity protein kinase, monopolar spindle 1 (Mps1) is one the main kinases of the spindle assembly checkpoint (SAC) critical for accurate segregation of sister chromatids during mitosis. A hallmark of cancer cells is chromosomal instability caused by deregulated cell cycle checkpoints and SAC dysfunction. Mps1 is known to be overexpressed in several solid tumors including triple negative breast cancer. Thus, Mps1 seems to be a promising target and small molecules targeting Mps1 entered clinical trials in solid tumors. ALL originates from malignant transformation of B-and T-lineage lymphoid precursors with a variety of genetic aberrations including chromosome translocations, mutations, and aneuploidies in genes responsible for cell cycle regulation and lymphoid cell development. While outcome is excellent for pediatric patients and younger adults, relapsed and refractory disease still remain a clinical challenge for elder patients. Here, we demonstrate for the first time preclinical efficacy of the small molecule Mps1 inhibitor (Mps1i) S81694 in T- and B- ALL cells including BCR-ABL1+-driven B-ALL. Materials and Methods: Expression of Mps1 was determined by RT-qPCR and WB in JURKAT, RS4-11 and BCR-ABL1+ cells (BV-173 and TOM-1). A small molecule Mps1i (S81694) was tested alone (0 to 1000nM) or in combination with imatinib, dasatinib, nilotinib and ponatinib in BCR-ABL1+ ALL cell lines. Cell viability and IC50 was assessed by MTS assays after exposure to Mps1i for 72h. In combination experiments, compounds were added simultaneously and relative cell numbers were determined at 72h with MTS assays and combination index (CI) values were calculated according to the Bliss model. Induction of apoptosis was evaluated by annexin-V exposure and PI incorporation at 72h with increasing doses of Mps1i. Cell-cycle distribution was determined by cytofluorometric analysis detecting nuclear propidium iodide (PI) intercalation at 48h. Phosphorylation of Mps1 was detected in synchronized (by nocodazole and MG-132) cells by immunofluorescence using an anti phospho-Mps1 antibody detecting Thr33/Ser37 residues. Time-lapse microscopy was used in cell lines in presence or absence of S81694 to determine mitosis duration. Bone marrow (BM) nucleated patient cells were obtained after informed consent and incubated in methylcellulose with cytokines with or without Mps1i for 2 weeks to determine colony growth. Results: Expression of Mps1 could be detected by RT-qPCR and at the protein level by WB in all cell lines (Figure 1A and B ). IC50 after Mps1i exposure alone was 126nM in JURKAT cells, 51nM in RS4-11 cells, 75nM in BV-173 cells and 83nM in TOM-1. Significant apoptosis as detected by phosphatidylserine exposure and PI incorporation in all cell lines with BCR-ABL1+ cell lines BV-173 and TOM-1 cells being the most sensitive (80% and 60% apoptotic cells respectively)(Figure 1C). Upon Mps1i exposure we observed targeted inhibition of Mps1 phosphorylation at Thr33/Ser37 residues indicating the specific on target effect of S81694 by inhibiting Mps1 autophosphorylation (Figure 1D and E). Cell cycle profile was generally lost after treatment with S81694 in all cell lines indicating aberrant 2n/4n distribution due to SAC abrogation (Figure 1F). Furthermore, we demonstrated that S81694 exposure accelerated significantly mitosis in BV-173 cell line from 36 minutes to 19 minutes indicating effective inhibition of SAC function (Figure 1G). Interestingly, S81694 induced significant apoptosis (70%) in the imatinib resistant BV173 cell line bearing the E255K-BCR-ABL1-mutation. Combination of S81694 with TKI imatinib, dasatinib and nilotinib (but not ponatinib) was strongly synergistic in BCR-ABL1+ cells (Figure 1H). Finally, we observed inhibition of colony formation in a patient with BCR-ABL1+ B-ALL after exposure to 100nM and 250nM S81694 (reduction of 85% and 100% respectively)(Figure 1I). Conclusion: Mps1i S81694 yields significant preclinical activity in T-and B-cell ALL including BCR-ABL1+ models. Interestingly S81694 was efficacious in a TKI resistant cell line. Disclosures Kaci: Institut de Recherches Internationales Servier (IRIS): Employment. Garrido:Institut de Recherches Internationales Servier (IRIS): Employment. Burbridge:Institut de Recherches Internationales Servier (IRIS): Employment. Dombret:AGIOS: Honoraria; CELGENE: Consultancy, Honoraria; Institut de Recherches Internationales Servier (IRIS): Research Funding. Braun:Institut de Recherches Internationales Servier (IRIS): Research Funding.

Whole Exome Sequencing of a Patient with Atypical Congenital Pure Red Cell Aplasia Has Enabled the Identification of a Novel Key Actor of Erythropoiesis That May be Involved in CDAI Physiopathology

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 3-4
Author(s):  
Elia Colin ◽  
Genevieve Courtois ◽  
Lydie Da Costa ◽  
Carine Lefevre ◽  
Michael Dussiot ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Homologous Recombination ◽  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Research Funding ◽  
Pure Red Cell Aplasia ◽  
Red Cell ◽  
Whole Exome

Background: The development of next generation sequencing techniques has brought important insights into the molecular mechanisms of erythropoiesis and how these processes can be perturbed in human diseases. This strategy may be valuable in some hereditary erythroid disorders where a subset of patients does not carry any mutations in the supposed causal gene and for which transgenic mouse models do not recapitulate the phenotype, suggesting that additional genetic events may be involved in pathogenesis. Here, we report the case of an adult patient presenting with atypical pure red cell aplasia associated with facial dysmorphy and chronic leg ulcers. Whole exome sequencing revealed a heterozygous missense mutation (R725W) in the CDAN1 gene, which has been previously reported in congenital dyserythropoietic anemia type I (CDAI). However, this mutation was also detected in her healthy brother, suggesting that this event alone was not sufficient to explain her phenotype. According to this hypothesis, we found an additional germline heterozygous nonsense mutation (Q732X) in the MMS22L gene, which was not shared by her unaffected relatives. MMS22L is a protein involved in homologous recombination-dependent repair of stalled or collapsed replication forks. Additionally, MMS22L is able to bind newly synthesized soluble histones H3 and H4 and exhibits a histone chaperone activity. MMS22L loading onto ssDNA during homologous recombination is promoted by the histone chaperone ASF1. Interestingly, CDAN1 acts as a negative regulator of ASF1 by mediating its sequestration in the cytoplasm, which results in the blocking of histone delivery. Aims: As MMS22L has never been reported in erythropoiesis before, we aimed to investigate the role of MMS22L in human erythropoiesis. Based on the data summarized above, the purpose of this study was also to determine the effect of combined inactivation of MMS22L and CDAN1 on in vivo erythropoiesis, while exploring the functional cooperation between both proteins. Results: To decipher the role of MMS22L in human erythropoiesis, we assessed the consequences of complete MMS22L inactivation in human cord blood CD34+ progenitors as well as in CD36+ immature erythroblasts using shRNA lentiviruses. This resulted in a severe decrease of cell proliferation and differentiation due to G1 cell cycle arrest, with a slight increase of apoptosis. Interestingly, this phenotype was not observed when MMS22L was inactivated in the granulo-monocytic lineage, in which differentiation was maintained, suggesting that erythroid cells, that are highly proliferative, are more sensitive to MMS22L inactivation. To better understand the effect of combined CDAN1 and MMS22L haploinsufficiency observed in the proband, we used zebrafish as an in vivo model. Mms22l and cdan1 expression were simultaneously or separately downregulated by about 50% using antisens morpholino oligomers. 48 hours later, zebrafish embryos were stained with o-dianisidine to detect hemoglobin-containing cells. We found that combined knock-down of mms22l and cdan1 resulted in severe anemia, while knock-down of mms22l or cdan1 alone did not lead to any erythroid disorder. This experiment provides a proof-of-concept, indicating that the phenotype of the proband is indeed caused by the combination of both MMS22L and CDAN1 mutations. Finally, in order to decipher the cooperation between MMS22L and CDAN1 we used the human erythroid UT-7 cell line. We found that CDAN1 inactivation resulted in a severe decrease in MMS22L expression within the nucleus, suggesting that CDAN1 may regulate MMS22L expression or localization. We therefore wanted to confirm these results by assessing MMS22L expression in B-EBV cell lines established from two CDAI patients with CDAN1 compound heterozygous mutations. We found a great decrease in MMS22L expression within the nucleus of the CDAI patients' cells when compared to three control B-EBV cell lines. Based on these results, we suggest that impairment of MMS22L trafficking to the nucleus could be involved in CDA1 physiopathology. Conclusion: Through comprehensive genetic analysis of a single case with atypical congenital anemia, we demonstrated for the first time that MMS22L, a cell cycle regulator, is essential for the process of erythropoiesis. The crosstalk between MMS22L and CDAN1 is currently under investigation and could bring important new insights into the physiopathology of CDAI. Disclosures Hermine: Novartis: Research Funding; Alexion: Research Funding; AB Science: Consultancy, Current equity holder in publicly-traded company, Honoraria, Patents & Royalties, Research Funding; Celgene BMS: Consultancy, Research Funding; Roche: Consultancy.

Sign in / Sign up

Export Citation Format

Share Document