Nodal/SMAD2 and Bone Morphogenic Protein 4 RNA Expression in Microgravity. Implications for Spacial Orientation Signaling in Hematopoesis and the Implications for the Hematosuppressive Effects of Space Flight

Abstract Introduction: Nodal proteins are members of the TGF alpha family which direct right left orientation in the developing embryo. Nodal/SMAD2 signaling controls BMP4 expression and with canonical Wnt signaling, regulates the posteriorization of the primitive streak, development of the mesoderm, development of early hematopoetic stem cells, and the property of self renewal in adult hematopoiesis. This study investigated the extent that microgravity alters expression of these spatially orienting proteins of BMP4, Nodal, Wnt, and SMAD2 thus influencing the anemia, thrombocytopenia, and immune alteration associated with space flight. Method: The Gene Expression Omnibus Database (GEO) of the National Institutes of Health was accessed to define RNA expression of Nodal, SMAD2, Wnt and BMP4 in hematopoetic cells in microgravity. Initial queries for "Low Gravity" studies resulted in 565 investigations within the GEO Omnibus. Of the 565 studies, the GSE136939, GSE101102, and the GSE101309 datasets were explored which assessed the lymphoid and macrophage cell lines TK6, U937, and Jurkat. RNA expression with normal and microgravity environments were compared. In the U937 cells hypergravity environments were also studied. Nodal, Lefty, Wnt, BMP4, as well as the NODAL signaling cascade proteins of SMAD 1/5, SMAD2, SMAD3, and the inhibitory SMAD 7. Affymetrix Expression Arrays were utilized for the TK^ and Jurkat cells assays. The U937 cell lines were assayed with NimbleGen Human Gene Assay Arrays. Results: Significant decreases in BMP4 expression were seen in microgravity environments (P value, <0.005) where SMAD2 expression was significantly elevated (P value <0.05) in microgravity. Conversely, hypergravity effects on U937 cells resulted in a reverse expression pattern. (BMP4 expression increased while SMAD2 decreased (P value <0.01) ). Comparisons of 14, 12, and 4 expression assays were made in U937, Jurkat, and TK6 cells respectively. LEFTY, NODAL, Wnt, SMA1/5/3/7 expression assays all showed changes in expression, but the small sample numbers resulted in poor statistical power and insignificant P values to detect differences in the RNA expression assessed. Comparisons of the gravitational pressures and time spent in these environments for the cellular cultures resulted in insignificant statistical differences. Conclusions: Microgravity induces decreases in BMP4 RNA expression and increases in SMAD2 RNA expression in TK6, U937, and Jurkat cell as defined by the GSE101309, GSE101102 and GSE136939 datasets. BMP4 is required for stem cell posteriorization, HSC number, and adult hematopoetic self renewal. This diminishment in BMP4 expression in microgravity may result in diminished HSC number and self renewal capacity. This qualitative change in gene expression in microgravity and may be a significant component of the anemia, thrombocytopenia, and immune alterations of space flight. Moreover such changes in RNA expression in microgravity implicates the importance of hematopoetic spacial orientation signals in normal hematopoiesis. Therapeutic targeting of the BMP/SMAD2 pathway in refractory multilineage hematopoetic diseases may prove to be fruitful. Statistical interpretation of the data is hindered by the small number of samples, the cell type, hematopoetic maturation stage of the samples, and microenvironmental differences in the studies utilized in these datasets. If this result is confirmed in future and larger studies, it would have significant biologic and therapeutic implications for long term space flight and the developing concept of spacial hematopoiesis. Disclosures No relevant conflicts of interest to declare.

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The Alteration of SEPT5 Gene Expression in BCR-ABL Positive Cells and Its Possible Correlation with the Development and / or Progression of Chronic Myeloid Leukemia (CML)

Blood ◽

10.1182/blood.v118.21.4415.4415 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4415-4415

Author(s):

Cintia Do Couto Mascarenhas ◽

Anderson Ferreira Cunha ◽

Ana Flavia Brugnerotto ◽

Sheley Gambero ◽

Joao Machado-Neto ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Cell Lines ◽

Real Time Pcr ◽

Blood Donors ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Blood Platelets ◽

Gene Expression Pattern ◽

P Value

Abstract Abstract 4415 The CML is a clonal disease of stem cells and its main feature is the unregulated production of a tyrosine kinase protein called BCR-ABL, the progression of the disease to accelerated phase or blast crisis may be associated with genomic instability. Because of this, the use of tools for the study of gene expression could bring new insights in the understanding of these mechanisms in the CML. In a recent study using SSH libraries, we compared the gene expression pattern between granulocytes of health control and CML patients, and we identified the gene SEPT5 expressed only in CML patients. Although the studies in the literature, there is not a clear relationship between the expression of this gene and the development or progression of CML. SEPT5 is a member of nucleotide binding proteins called septins that were firstly described in yeast as cell division cycle regulatory proteins. This gene was reported in patients with AML translocated with MLL gene, in adult human brain and heart; it is also associated with alpha granules of human blood platelets. The aims of this study are to carry a functional analysis of SEPT5 in differents cells line and to study the relationship of this gene and the development and/or progression of CML. The gene expression evaluation was made in granulocytes, mononuclear cells and total leukocytes of CML patients and healthy blood donors in peripheral blood. It was also evaluated in bone marrow donors, in human cell lines (K562, HL60 and NB4) and in mice cell lines (BaF3/BCR-ABLp210 and BaF3T315I), performed by real-time PCR for the following genes: SEPT5, β-actin and GAPDH. Experiments were also performed to verify the difference between the chemotaxis of granulocytic cells from controls and patients by ELISA. Data were analysed statistically using the ANOVA followed by Dunnett’s test – P value of less than 0.05 was considered to be significant. The study was approved by the Research Ethic Committee of the Faculty of Medical Sciences of University of Campinas. The gene expression of SEPT5 was evaluated by real time PCR using the same samples used in the library construction to validate the results found in the SSH library. The data confirmed our previous results, showing that the SEPT5 expression is increased in all cells of patients compared to controls. The same results were observed when we studied the expression comparing individually patients and health blood donors, suggesting that this protein could be increased in all human cells that present the translocation BCR-ABL. The level of expression of this gene in HL60 and NB4 was significantly lower than in K562 cell line. The experiments with mice cell lines showed a higher expression of this gene in BaF3T315I when compared to BaF3BCR-ABLp210. We obtained a significant expression difference in all experiments (p <0.05). The spontaneous and stimulated with IL-8 chemotaxis assays used granulocytes and were assessed using chamber containing 96 wells. However, although the results suggest an increased chemotactic activity in patients, there were no significant differences (p<0.05) between controls and patients – regardless of whether the chemotaxis was spontaneous or stimulated with IL-8. In mammals the SEPT5 gene is associated with cellular processes such as exocytosis, apoptosis, leukemogenesis, carcinogenesis and neurodegeneration. Therefore, molecules capable of interacting with the septins, either at biochemical or molecular level, can bring information about their functions in cytokinesis. Studies indicate that the human septins can interact among themselves and with other components of the cytoskeleton – this may be a relevant observation regarding the function of this gene in cancer. The SEPT5 can be activated by different pathways – this may increase expression in translocated cells. Despite major advances in the treatment of CML, the treatments available are not capable of inactivating all the signaling pathways activated by BCR/ABL. Our results demonstrate that SEPT5 may be involved in the pathophysiology of CML. Also, it is clear the importance of the study of pathways that could culminate in its high expression or the triggering of other unknown pathways involved in the development of CML. The increased expression of this gene may be related to disease progression, and finally, the identification of several important genes may lead to a better understanding of CML and helping to identify new therapeutic targets. FAPESP/INCT. Disclosures: No relevant conflicts of interest to declare.

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PLAG1 and USF2 Regulate Primitive Hematopoietic Expression of Musashi-2

Blood ◽

10.1182/blood.v126.23.3583.3583 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3583-3583

Author(s):

Muluken S Belew ◽

Stefan Rentas ◽

Laura de Rooij ◽

Kristin J Hope

Keyword(s):

Gene Expression ◽

Stem Cell ◽

Cell Lines ◽

Regulatory Elements ◽

Hematopoietic Cell ◽

Minimal Promoter ◽

Luciferase Reporter ◽

Specific Expression ◽

Self Renewal ◽

Model Cell

Abstract The Musashi-2 (MSI2) RNA binding protein is now recognized as a key regulator of hematopoietic stem cells (HSCs). Its expression is most elevated in the primitive HSC compartment and progressively decreases with differentiation. In mouse models of CML, ectopic expression of MSI2 drives progression from the chronic to the blast crisis state while in the human context its aberrantly high expression correlates with more aggressive CML disease states and is associated with poor prognosis in AML. These studies suggest that the precise molecular regulation of MSI2 gene expression may be among the critical mechanisms underlying balanced HSC self-renewal and differentiation and as a result, the prevention of leukemic transformation/progression. Despite the clear importance of understanding how Msi2 maintains an appropriate stem cell-specific expression level, very little is understood of the transcription factors (TFs) that mediate this. To define those factors that govern MSI2 expression and function specifically in the HSC compartment we undertook a systematic approach to map and define relevant regulatory elements of the MSI2 minimal promoter. We dissected a 3.5 kb region 5' upstream of MSI2's translational start site (TSS) shared between mouse and human and thus having the greatest potential of containing regulatory elements key to a conserved MSI2 stem-cell-specific gene expression program. Progressive 5'-terminal deletions of this region cloned upstream of a luciferase reporter gene and transfected into K562 and 293T model cell lines allowed us to define a minimal conserved promoter region from -588 to -203 bp upstream of the TSS that reports accurately on endogenous MSI2 expression. Coupled with in silico prediction of TF that bind this region, systematic TF binding site mutagenesis and luciferase reporter assays in model cell lines identified USF2 and PLAG1 as TFs whose direct binding to the MSI2 minimal promoter direct reporter activity. Loss and gain of function studies in K562 cells confirm that these factors co-regulate the transactivation of endogenous MSI2. Moreover we show in the most relevant primary human CD34+ hematopoietic cell context that these factors bind the MSI2 minimal promoter. While USF2 is a ubiquitously expressed TF across the hematopoietic hierarchy, the uniquely restricted expression of PLAG1 within only the most primitive of hematopoietic cells suggests that it specifically contributes to the heightened stem cell-specific expression of MSI2. Consistent with its role as a key driver of MSI2 and thus an enforcer of its pro-self-renewal functions, we found that overexpression of PLAG1 in human Lin-CD34+ cord blood cells enhanced MSI2 transcription and increased total Colony Forming Unit (CFU) output and re-plating efficiency of primitive CFU progenitors. PLAG1 overexpression also offered a pro-survival advantage to these cells as evidenced by a more than two-fold reduction in Annexin V positive cells compared to negative controls. We have thus described important transcriptional circuitry that governs stem-cell specific expression of MSI2 while at the same time functionally validated PLAG1 as a novel factor capable of modulating primitive hematopoietic cell self-renewal and survival. Disclosures No relevant conflicts of interest to declare.

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Identification of synthetic lethal interactions with the BRAF oncogene in colorectal cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.4_suppl.403 ◽

2013 ◽

Vol 31 (4_suppl) ◽

pp. 403-403

Author(s):

Loredana Vecchione ◽

Valentina Gambino ◽

Giovanni d'Ario ◽

Sun Tian ◽

Iris Simon ◽

...

Keyword(s):

Gene Expression ◽

Cell Lines ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Gene Expression Signature ◽

Gene Signature ◽

Braf V600e ◽

P Value ◽

Synthetic Lethal ◽

Braf Mutant

403 Background: Approximately 8-15% of colorectal (CRC) patients carry an activating mutation in BRAF. This CRC subtype is associated with poor outcome and with resistance, both to chemotherapeutic treatments and to tailored drugs. We recently showed that BRAF (V600E) colon cancers (CCs) have a characteristic gene expression signature (1, 2) which is found also in subsets of KRAS mutant and KRAS-BRAF wild type (WT2) tumors. Tumors having this gene signature, referred as “BRAF-like”, have a similar poor prognosis irrespective of the presence of the BRAF (V600E) mutation. By using a shRNA-based genetic screen in BRAF mutant CC cell lines we aimed to identify genes and pathways necessary for survival and growth of BRAFmutant CC. Such studies may reveal additional targets for therapy and potentially provide new biomarkers for patient stratification Methods: We identified 363 genes that are selectively overexpressed in BRAF mutant tumors as compared to WT2 type tumors, based on gene expression profiles of the PETACC3 (1) and Agendia (2) datasets. The TRC human genome-wide shRNA collection (TRC-Hs1.0) was used to generate a 1815 hairpins sub-library targeting those identified genes (BRAF library). BRAF(V600E) CC cell lines were infected with the BRAF library and screened for shRNAs that cause lethality. LIM1215 CC cell line (WT2) was used as a control. Cells stably expressing the shRNA library were cultured for 13 days, after which shRNAs were recovered by PCR. Deep sequencing was applied to determine the specific depletion of shRNA in BRAF(V600E) cells as compared to LIM1215 cells Results: Candidate genes were identified by using following filtering criteria: depletion in BRAF(V600E) cells by at least 50% and depletion in BRAF(V600E) cells 1, 5-fold higher than in control cells with the corresponding p-value to be ≤ 0.1. A total of 34 genes met our criteria of which 6 genes were presented with more than one hairpin and were concordant across the cell lines selected for validation. Conclusions: We identified candidate synthetic lethal genes in BRAF mutant CC cell lines. Functional analysis is ongoing. Data will be presented. References 1. J Clin Oncol 2012 Apr 20;30(12):1288-9 2. Gut (2012). doi:10.1136/gutjnl-2012-302423

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Engineered Bcor Mutations Lead to Acute Lymphoblastic Leukemia of Progenitor B-1 Lymphocyte Origin in a Sensitized Background

Blood ◽

10.1182/blood-2018-99-115388 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1331-1331

Author(s):

Mianmian Yin ◽

Yang Jo Chung ◽

R. Coleman Lindsley ◽

Yeulin Zhu ◽

Robert L. Walker ◽

...

Keyword(s):

Gene Expression ◽

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Cell Lines ◽

Lymphoblastic Leukemia ◽

Regulatory Elements ◽

Leukemic Transformation ◽

Self Renewal ◽

Hematopoietic Stem ◽

Indel Mutation

Abstract Chromosomal translocations resulting in NUP98 fusion genes have been associated with a wide spectrum of hematologic malignancies, including MDS, AML, T-ALL, and B cell precursor (BCP) ALL. Based on gene expression profiles and murine transplantation experiments, it is thought that NUP98 fusions can confer aberrant self-renewal potential to hematopoietic cells. Approximately 90% of mice that express a NUP98-PHF23 (NP23) fusion in the hematopoietic compartment, under the control of Vav1 regulatory elements develop AML and/or T-ALL. However, approximately 10% of NP23 mice develop an aggressive acute lymphoblastic leukemia of B1-lymphocyte progenitor origin (pro B-1 ALL). Whole exome sequencing demonstrated that all NP23 pro-B1-ALL had acquired somatic frameshift mutations of the BCL6 co-repressor (Bcor) gene, and most had acquired mutations in the Jak/Stat pathway. To determine whether experimentally engineered Bcor mutations would lead to pro B-1 ALL, we used CRISPR-Cas9 to introduce Bcor indel mutations into NP23 hematopoietic stem and progenitor cells through the use of Bcor single guide RNAs (Bcor sgRNA). Recipient mice transplanted with NP23 bone marrow (BM) or fetal liver (FL) cells that had been transduced with a Bcor sgRNA developed pro B-1 ALL, characterized by a B-1 progenitor immunophenotype, clonal Igh gene rearrangement, and Bcor indel mutation, whereas control recipients did not. In addition, similar to some human BCP ALL, the Bcor sgRNA/NP23 murine pro B-1 ALL had acquired somatic mutations in Jak kinase genes. A distinct subset of pediatric BCP ALL are characterized by rearrangement and overexpression of the CRLF2 gene (designated CRLF2r); the CRLF2 gene is the receptor for thymic stromal lymphopoietin (TSLP), a cytokine that plays a role in normal progenitor B1 cell development. The NP23 pro-B1 ALL are similar to CRLF2r BCP ALL in terms of a preferential V heavy chain (VH) usage, gene expression profile, and propensity for acquired JAK/STAT pathways mutations. JAK inhibitors (ruxolitinib and tofacitinib) induced apoptosis and inhibited the growth of pro B-1 ALL cell lines established from Bcor sgRNA/NP23 recipients, at clinically achievable concentrations (10-100 nM). Taken together, these findings demonstrate that a CRISPR-induced Bcor frameshift collaborates with an NP23 transgene to predispose B-1 progenitors to leukemic transformation. These two events are unlikely to be sufficient for leukemic transformation, as we detected spontaneous Jak pathway mutations that were required for continued growth of the leukemic cells. This constellation of mutations (NP23 expression leading to increased stem cell self-renewal, Bcor frameshift leading to impaired B cell differentiation, and Jak pathway mutations leading to dysregulated proliferation) is similar to that seen in human BCP ALL patients, and suggests that the NP23/Bcor mutant mice and cell lines will be a useful model for human pro-B1 ALL. Disclosures Aplan: NIH Office of Technolgy Transfer: Employment, Patents & Royalties: NUP98-HOXD13 mice.

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Abstract 17064: Enrichment of Cardiotoxic Gene Expression Profile in Human Induced Pluripotent Stem-Cell Derived Cardiomyocytes in Response to Tyrosine Kinase Inhibitor Nilotinib

Circulation ◽

10.1161/circ.138.suppl_1.17064 ◽

2018 ◽

Vol 138 (Suppl_1) ◽

Author(s):

Praful Aggarwal ◽

Matthew White ◽

Andrea Matter ◽

Amy Turner ◽

Benjamin Olson ◽

...

Keyword(s):

Gene Expression ◽

Tyrosine Kinase ◽

Gene Expression Profile ◽

Cell Lines ◽

Expression Profile ◽

Molecular Mechanisms ◽

P Value ◽

Significant Differential Expression ◽

Cell Index ◽

Beat Rate

Small molecule tyrosine kinase inhibitors (TKIs) are a valuable class of therapeutics with widespread clinical utility against multiple cancers. However, there is strong evidence that TKIs are associated with cardiotoxicity and adverse cardiovascular events. Our understanding of the underlying mechanisms related to TKI induced cardiotoxicity is limited. Human iPSC derived cardiomyocytes (hiPSC-CMs) provide a flexible platform and unique model to study the underlying molecular mechanisms associated with TKI associated cardiotoxicity. In this study we describe the gene expression profile between hiPSC-CM cell lines which exhibit susceptibility vs. resistance. RNA-seq analysis was performed in hiPSC-CM cell lines from six participants in the NHLBI HyperGEN study (A to F). Experiments were performed in triplicate using sunitinib (SUN), vandetanib (VAN), gefitinib (GEF) and nilotinib (NIL). We analyzed beat rate, cell index and ATP viability as physiological measurements of CM toxicity and defined a 20% change from the normalized control as TKI susceptibility. Differential gene expression analysis was performed using DESeq2. We observed significant physiological differences between the different hiPSC-CMs after TKI treatment (beat rate, cell index and ATP viability). The most variable cell index and beat rate response was observed for NIL. Based on cell index, lines B, D, E were resistant while A, C, F were significantly more susceptible to NIL. Principal component analysis showed that the variance in gene expression was the highest after NIL treatment when compared to controls (16% for NIL; 11% for VAN; 6% for SUN and 5% for GEF). A total of 567 genes exhibited significant differential expression changes (adj. p-value ≤ 0.1) after NIL treatment in susceptible versus resistant lines. Pathway analysis showed significant enrichment for cardiotoxicity including pathways implicated in cardiac infarction, fibrosis, hypertrophy, and congestive cardiac failure. Taken together, our results identify unique gene expression changes associated with TKI cardiotoxicity. Furthermore, the variability in TKI susceptibility between different hiPSC-CM lines highlights the need to comprehensively assess cardiotoxicity in a diverse set of lines on a physiological and molecular level.

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Generating AML-Specific Peripheral Blood Autologous Cytotoxic T-Lymphocytes (CTLs)

Blood ◽

10.1182/blood.v124.21.5806.5806 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5806-5806

Author(s):

Rohtesh S. Mehta ◽

Xiaohua Chen ◽

Antony Jeyaraj ◽

Paul Szabolcs

Keyword(s):

Gene Expression ◽

T Cells ◽

T Cell ◽

Gene Expression Profile ◽

Cell Lines ◽

Expression Profile ◽

Leukemia Cell ◽

P Value ◽

Cell Gene Expression ◽

Cell Gene

Abstract Background: Ex-vivo expansion of CBT-cells using CD3/CD28 co-stimulatory beads, IL-2 + IL-7 and subsequent priming against leukemia cell lines using IL-15 generated specific CTLs. [1, 2] Hypothesis: We hypothesized that (a) patient-derived AML-specific PB auto CTLs could be generated with immune-stimulatory culture condition (b) Resistant AML samples would possess gene expression profiles similar to MDSCs (myeloid-derived suppressor cells) (c) Frequency of Tregs (CD4+CD25brightFoxP3+) and T-cell co-signaling molecules gene expression will be different between effective and ineffective CTLs. Methods: AML & auto T-cells were purified from cryopreserved PBMC of AML patients admitted with acute blast crisis (n=8). AML blasts were sustained in StemSpan™ Serum-Free media [STEMCELL Technologies] with MSC support + cytokine cocktail (IL-3, SCF, FLT3L, GMCSF, IL-4). T-cells were expanded in culture for 2 weeks as reported [1, 2] and subsequently primed with γ-irradiated auto AML weekly X 3 with IL15 + CD28ab [BD Biosciences]. At the end of week 3 (EOW3), cytotoxicity was assessed against AML and irrelevant targets - IM9 (lymphoid) and U937 (myeloid) cell lines, loaded with BATDA at an E:T ratio of 40:1, 20:1, 10:1 and 5:1 using DELFIA® EuTDA assay.[2] IFN-γ ELISPOT assay against same targets was also done.[2] RT-qPCR analysis was performed on AML & T-cells before and after priming, using Power SYBR® Green master mix (Thermo Fisher Scientific) and StepOne Plus system [Life Technologies]. Two-tailed student t-testcompared experimental groups. Results · T-cells expanded in all samples (n=8) with a median expansion of 155-fold (range 11-489), at EOW3. · ELISPOT assay was positive in 4/8 samples. [Fig 1] · CTL assay was difficult to standardize for primary AML blasts due to high degree of spontaneous apoptosis (>30% spontaneous release [SR]). · 2/8 samples were deemed evaluable (SR<30%). · Both samples showed AML-specific lysis. [Fig 2] · Overall, AML-specific autologous CTL could be generated from 5 of 8 samples based on ELISPOT & CTL assays, regardless of original FAB immunophenotype, not shown. · Tregs proportion declined significantly in effective CTLs post-priming as compared to pre-priming (56% to 24%, p-value 0.046, n=4). [Fig 3] · T-cell gene expression profiling showed significant differences in effective vs ineffective CTLs. [Table 1] · Resistant AML (n=3) had up-regulated downstream markers associated with MDSC generation compared to “non-resistant” AML (n=5). [Table 2] Conclusions (a) AML-specific auto CTLs can be generated (b) Tregs decreased with priming in effective CTLs (c) differential T-cell gene expression profile exists between effective and ineffective CTLs (d) AML gene expression suggests MDSC-like profile in resistant samples.Abstract 5806. TABLE 1:T-CELL GENE EXPRESSION PROFILE (POST VS PRE-PRIMING)Effective CTLs (n=5)Ineffective CTLs (n=3)GeneΔΔ Ct(Post - Pre) (mean, SEM)P-valueFold change (mean, SEM)ΔΔ Ct(Post - Pre) (mean, SEM)P-valueFold change (mean, SEM)4-1BB-3.17 (0.76)0.02514 (7.7)1.98 (1.04)0.190.39 (0.22)HVEM-2.43 (0.61)0.0287.3 (3.7)0.14 (1.65)0.951.57 (1.28)LIGHT-3.62 (0.73)0.01617.3 (7.3)1.78 (1.84)0.441.1 (0.98)PRKC-α-2.03 (0.47)0.0234.6 (1.1)1.89 (0.36)0.0340.29 (0.08)PRKC-θ-3.36 (0.59)0.0113.7 (6.7)0.25 (0.59)0.710.99 (0.41)LAIR1-3.81 (0.42)0.00316.2 (5.6)-1.35 (2.20)0.6017.15 (16.5)PP2A-2.40 (0.57)0.0256.7 (2.6)0.49 (1.57)0.791.89 (1.52)2B4-1.53 (1.14)0.274.98 (1.82)-3.48 (0.11)0.0211.2 (0.9)LTA-α-1.18 (0.78)0.233.61 (2.11)2.69 (0.18)0.0430.16 (0.02)LTA-β-0.93 (0.63)0.242.49 (0.99)2.24 (0.47)0.0420.23 (0.08) TABLE 2: GENE EXPRESSION PROFILE RESISTANT VS NON-RESISTANT AML Gene ΔΔ Ct (mean, SEM) 95% CI P-value Relative fold change JAK1 -4.63 (1.98) -9.48 0.21 0.0579 24.83 JAK2 -5.38 (0.94) -7.67 -3.08 0.0012 41.52 JAK3 -5.90 (2.17) -12.81 1.01 0.0726 59.77 S100A8 -7.16 (2.66) -14.01 -0.32 0.0432 143.27 S100A9 -8.31 (2.75) -15.04 -1.59 0.0233 318.37 c-myc -2.78 (0.59) -4.24 -1.33 0.0034 6.89 Refs: 1.Davis et al. Cancer Research 2010;70(13):5249 2.Jeyaraj A, Chen X, Szabolcs P. IL-15 Induced Polyclonal CTL Generated From Expanded CBT Cells Against Leukemia Cell Lines Constitutes IFN-γ Producing Cells and TCRγδ Cells. ASH 2012 Annual Meeting Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.

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Hyperbaric Oxygen Increases Sensitivity to Chemotherapy in Acute Leukemia

Blood ◽

10.1182/blood.v126.23.1399.1399 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1399-1399

Author(s):

Tara L. Lin ◽

Amanda Wise ◽

Abigale Berry ◽

Joseph Fontes ◽

Partha Kasturi ◽

...

Keyword(s):

Gene Expression ◽

Gene Expression Profiling ◽

Cell Lines ◽

Expression Profiling ◽

Chemotherapy Resistance ◽

Leukemia Cells ◽

P Value ◽

Significant Challenge ◽

Increased Sensitivity

Abstract Introduction: Chemotherapy resistance remains a significant challenge in the treatment of adults with Acute Myeloid Leukemia (AML). Although new targets and novel agents are under clinical investigation, the majority of AML patients are treated with conventional induction chemotherapy consisting of an anthracycline and cytarabine, with little modification in over 40 years. Insights into the biology of resistance to cytarabine and anthracyclines may suggest novel strategies in the treatment of AML. Hyperbaric oxygen (HBO) treatment is commonly used in the treatment of decompression sickness from scuba diving, wound healing and infections. 100% oxygen is administered at increased pressure. Limited published data suggest a role for HBO to cause in vitro apoptosis of leukemia cell lines, but no mechanism has been described. We examined the role of HBO in AML sensitivity to chemotherapy, reactive oxygen species (ROS) generation, glycolytic pathway activation and gene expression. Methods: Human AML cell lines HL-60 and MV-411 were treated with escalating doses of cytarabine or daunorubicin as single agents followed by HBO treatment for 2 hours or normobaric conditions. HBO treatment consisted of exposure to 100% oxygen at 2.5 atmosphere absolutes for a total of 2 hours in a specifically designed in vitro HBO chamber. At 24 hours after chemotherapy dosing, leukemia cells were analyzed for proliferation using Cell Titer 96 Aqueous MTS assay (Promega). Apoptosis was measured using Annexin V-FITC and analyzed by flow cytometry. In order to understand potential mechanisms of HBO activity on leukemia cells, studies of HBO-treated cells without use of chemotherapy were also performed. CellROX Oxidative Stress assay (Life Technologies) was performed at 24 hours. CellROX Green and Deep Red reagents measured ROS level in the DNA and cytoplasm. RNA was isolated and real-time PCR was performed using CFX384 Real-time PCR system using primer sets specific for human hexokinase2 and phosphofructokinase to examine the effects of HBO on glycolysis. Gene expression profiling was performed using the Human Transcriptome Array 2.0 (Affymetrix) and examined differences in gene expression following HBO treatment of HL-60 cells and bone marrow from an AML patient. Results: HBO-treated leukemia cells had increased sensitivity to cytarabine (Table 1, Figure 1) (MV411 response to cytarabine: IC50 2649nM without HBO treatment, 1921nM with HBO, p=0.02; HL60 response to cytarabine: linear response without HBO, IC50 1625nM with HBO, p<0.035). Decreased proliferation was also seen with HBO-treated cells in response to daunorubicin but was not statistically significant (Table 1). Similarly, apoptosis from cytarabine in HL-60 cells was increased with HBO treatment (p<0.02), but the results with daunorubicin were not significant. Significant increases in ROS levels were observed at 24 hours post-HBO for both cell lines when compared to non-treated cells (HL60 DNA p<0.0001, HL60 cytoplasm p<0.01, MV411 DNA p<0.01, MV411 cytoplasm p<0.01). Glycolysis pathway components human hexokinase 2 and phosphofructokinase were upregulated in HL60 and MV411 cells treated with HBO compared to controls. Gene expression profiling showed that approximately 200 genes in coding regions were upregulated in HBO-treated HL-60 cells; specific piRNA clusters were downregulated in both AML cell line HL60 and a bone marrow sample from a patient with relapsed AML when treated with HBO. Conclusions: HBO treatment significantly increases the sensitivity to cytarabine AML cell lines in vitro. Increased ROS, upregulation of glycolysis-related enzymes and changes in gene expression profiling provide insight into the potential mechanisms of HBO enhanced chemotherapy sensitivity. Given the safety and commonplace use of HBO for other indications, and the significant challenge of chemotherapy resistance, it is important to pursue further studies to understand the biology of HBO treatment in AML. Table 1. IC50 (nM) of chemotherapy agents in AML cell lines with and without co-treatment with HBO. Cytarabine Daunorubicin no HBO HBO p-value no HBO HBO p-value MV411 2649 1921 0.0214 709.1 641.1 ns HL60 * 1625 0.0346 639 406.5 ns * linear response, no IC50 available Figure 1. Increased sensitivity to cytarabine in AML cell lines following treatment with HBO. Figure 1. Increased sensitivity to cytarabine in AML cell lines following treatment with HBO. Disclosures No relevant conflicts of interest to declare.

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Genomic Characterization of in Vitro Acquired-Resistance to Proteasome Inhibitors

Blood ◽

10.1182/blood-2021-148584 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2651-2651

Author(s):

Hugues De Boussac ◽

Alboukadel Kassambara ◽

Amelie Machura ◽

Djamila Chemlal ◽

Claire Gourzones ◽

...

Keyword(s):

Gene Expression ◽

Drug Resistance ◽

Board Of Directors ◽

Cell Lines ◽

Expression Profiling ◽

Proteasome Inhibitors ◽

Resistance Mechanisms ◽

P Value ◽

Glycolytic Metabolism ◽

Advisory Committees

Abstract Multiple myeloma (MM) is the second most common hematological malignancy characterized by an abnormal clonal proliferation of malignant plasma cells. Despite the introduction of novels agents that have significantly improved clinical outcomes, MM patients invariably relapse. A better understanding of the drug resistance mechanisms and development of biomarkers remain of major interest to improve the treatment of patients. In order to further investigate the mechanisms involved in resistance to proteasome inhibitors (PI), we have derived and characterized PI-resistant human myeloma cells lines (HMCLs) from different molecular subgroups including XG2BR t(12;14), XG7BR t(4;14), XG19BR t(14;16), and XG1BR t(11;14). These cell lines were cultured continuously with escalating concentrations of Bortezomib (Btz) during 12 months and showed a significant resistance to Bortezomib compared to their parental cell lines (mean IC50: Btz-resistant HMCLs =5.5nM vs parental HMCLs=2.5nM, p<0.05). Of interest, we demonstrated that Btz-resistant HMCLs are also significantly more resistant to Carfilzomib (Cfz) and Ixazomib (Ixa) PIs (mean IC50: Btz-resistant HMCLs =6nM for Cfz and =70nM for Ixa vs parental HMCLs=3nM, for Cfz, p<0.05 ; and =21nM for Ixa, p<0.05. No significant cross-resistance was observed with other therapeutic agents including melphalan, dexamethasone and IMIDs indicating that the observed drug resistance mechanisms are specifically related to PIs. In order to understand the PIs resistance mechanisms acquired by MM cells, we used a combination of genomic approaches including whole genome sequencing, and comparative transcriptomic analysis. Among the 40 mutations identified in Btz-resistant HMCLs compared to the parental ones, a mutation residing in the Btz-binding pocket in the proteasome beta5-subunit (PSMB5) gene was identified. This mutation has already been found in other models of PIs-resistant MM cell lines and in relapsed MM patients, and is associated to PIs- resistance by reducing the PI binding capacity and impairing the chemotrypsin-like catalytic activity of the 20S proteasome. When we compared the gene expression profiling of Btz-resistant HMCLs with the parental ones, we identified a gene expression signature significantly enriched in nucleotide excision repair (NER) pathway with an increased expression of ERCC1, ERCC5, LIG1, POLD1 in the Btz-resistant HMCLs (FC >1.5). Since, protein ubiquitination is essential in regulation and coordination of various pathways of DNA damage recognition, signaling and repair, proteasome inhibitors affect DNA repair, overexpression of DNA repair pathways may participate in drug resistance mechanisms. Using gene expression profiling data, we also identified a significant downregulation of 8 solute carrier protein (SLC) intake transporters (SLC6A6, SLC16A1, SLC16A14, SLC16A10, SLC25A13, SLC5A6, SLCO3A1, SLCO4A1) together with a significant upregulation of xenobiotic receptors (RXRA, RXRB) in Btz-resistant HMCLs compared to parental HMCLs (FC> or < 1.5; p value <0.05). In addition, several genes involved in antioxidant response (NQO1), and in glutathione regulation (MGST1, MGST2, GSTO1) were also overexpressed in Btz-resistant HMCLs (FC>1.5 p value <0.05). Investigating the deregulated genes involved on energy metabolism that is often associated with resistance, we identified an upregulation of glycolytic enzymes directly involved in glycolytic metabolism (ALDOC, ENO3, HK1, PDK1, PDK3, PFKB3, PFKB4, PFKL, SLC2A1 (FC>1.5 p value <0.05) in the Btz-resistant HMCLs. Altogether our data underline a significant deregulation of genes involved in cell metabolism and drug clearance system that allow the PI resistant-MM cells to maintain metabolic homeostasis and survival in stringent redox conditions. This is in accordance with described mechanisms linking drug resistance and glycolytic metabolism in cancers cells. Metabolomic analyzes are currently ongoing for functional validation. Altogether, drug-resistant cell lines represent an attractive preclinical model to test molecules targeting these pathways in order to identify new therapeutic strategies to overcome PI resistance in MM. Disclosures De Boussac: Diag2Tec: Current Employment. Kassambara: Diag2Tec: Consultancy. Machura: Diag2Tec: Current Employment. Chemlal: Diag2Tec: Current Employment. Vincent: Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees. Herbaux: Abbvie: Honoraria, Research Funding; Roche: Honoraria; Janssen: Honoraria; Takeda: Honoraria, Research Funding. Bruyer: Diag2Tec: Current Employment. Moreaux: Diag2Tec: Consultancy.

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Quantitative Proteomic Analysis of Relapsed Multiple Myeloma Identifies CDK6 Upregulation As a Potential Targetable Resistance Mechanism for Lenalidomide

Blood ◽

10.1182/blood-2019-130682 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2567-2567

Author(s):

Yuen Lam Dora Ng ◽

Stephan Bohl ◽

Evelyn Ramberger ◽

Oliver Popp ◽

Imke Bauhuf ◽

...

Keyword(s):

Multiple Myeloma ◽

Protein Expression ◽

Cell Lines ◽

Myeloma Cell ◽

P Value ◽

Rna Expression ◽

Myeloma Cells ◽

Multiple Myeloma Cell ◽

Protein Levels ◽

Pre Treatment

Lenalidomide, an immunomodulatory drug (IMiD), is highly active and broadly used for the treatment of multiple myeloma. Despite high initial remission rates, patients frequently relapse and become resistant to the drug. Comprehensive analyses of gene mutations and RNA expression have identified inactivating mutations and RNA downregulation in cereblon (CRBN), the primary target of lenalidomide, in some of the resistant patients. However, the underlying resistance mechanism for the majority of cases remains unknown. Here, we performed quantitative tandem mass tag (TMT)-based proteomic analyses and RNA sequencing in five paired pre-treatment and relapse samples from multiple myeloma patients treated with drug combinations comprising lenalidomide to identify changes in protein expression associated with resistance. Using a stringent cut-off with an adjusted P value < 0.1 and log2 fold change (FC) > 2, we found 7 proteins to be significantly upregulated and 10 proteins to be downregulated in the relapsed versus pre-treatment multiple myeloma samples. Of these 17 deregulated proteins at relapse, only two were also found to be deregulated on the RNA expression level (adjusted P value < 0.1) as assessed by RNA sequencing. In general, correlation between protein expression levels and RNA expression levels were weak (median Pearson correlation coefficient r=0.35). Among the top upregulated proteins in relapse samples was cyclin-dependent kinase 6 (CDK6) with an average log2 FC of 2.1. Protein and RNA levels of CDK6 showed only weak correlation (r=0.4) and CDK6 RNA was not differentially expressed between the relapse and pretreatment samples. To validate the findings of the proteomic analysis, we assessed CDK6 protein levels by western blot in additional patient samples obtained at diagnosis (N=4) and at relapse (N=9). This confirmed a high CDK6 protein expression in 6 of 9 relapse samples while CDK6 could not be detected in the 4 pre-treatment samples. In order to determine the impact of CDK6 on drug sensitivity, we overexpressed CDK6 using either a retro- or lentiviral vector system in multiple myeloma cell lines. In two multiple myeloma cell lines tested, MM.1S and OPM2, CDK6 overexpression reduced sensitivity to lenalidomide and pomalidomide, but not to melphalan, bortezomib, or dexamethasone. To examine whether lowered IMiD-sensitivity can be overcome by CDK6 inhibition, we treated multiple myeloma cell lines either with the CDK6 inhibitor palbociclib, an IMiD-based CDK6-selective proteolysis targeting chimera (PROTAC) or a non-selective CDK6-PROTAC which is also capable of pomalidomide-mediated degradation of IKZF1 and IKZF3 (Brand et al., Cell Chem Biol 2019). Both palbociclib and CDK6-selective PROTAC as single treatments had only mild effects on the majority of multiple myeloma cells, implying that multiple myeloma cells are generally not dependent on CDK6. In contrast, the combination treatment of palbociclib with lenalidomide, or the non-specific CDK6/IKZF1/IKZF3-targeting PROTAC significantly inhibited proliferation, producing synergistic effects on the decrease of myeloma cell viability in 6 multiple myeloma cell lines, including those with a low IMiD sensitivity like RPMI-8226 and L363. This demonstrates that CDK6 inhibition or degradation enhances the cytotoxic effects of IMiDs. In order to investigate a potential mechanism for the synergistic effects of CDK6 inhibition and IMiDs, we analyzed protein levels in treated cells. CDK6 inhibition or degradation had no effect on CRBN protein levels nor on lenalidomide-induced degradation of IKZF1 and IKZF3. In contrast, combined degradation of CDK6, IKZF1, and IKZF3 revealed decreased protein levels of c-MYC, which was not observable in cells treated with palbocicilib, CDK6-selective PROTAC or pomalidomide alone. In conclusion, quantitative proteomics in primary multiple myeloma samples identified new druggable candidates including CDK6 in relapse that were overlooked by RNA expression analyses. Inhibition of CDK6 by palbociclib or a PROTAC sensitizes multiple myeloma cells to IMiDs and results in synergism when used in combination. Disclosures Bohl: Pfizer: Honoraria. Bullinger:Menarini: Honoraria; Novartis: Honoraria; Pfizer: Honoraria; Sanofi: Honoraria; Seattle Genetics: Honoraria; Janssen: Honoraria; Jazz Pharmaceuticals: Honoraria; Amgen: Honoraria; Astellas: Honoraria; Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Daiichi Sankyo: Honoraria; Gilead: Honoraria; Hexal: Honoraria; Bayer: Other: Financing of scientific research; Abbvie: Honoraria. Kroenke:Celgene: Consultancy, Honoraria; Takeda: Consultancy.

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Development of a Gene Expression Panel, for the Prediction of Protein Abundances in Cancer Cell Lines

Current Bioinformatics ◽

10.2174/1574893616666210517162530 ◽

2021 ◽

Vol 16 ◽

Author(s):

Gunhee Lee ◽

Yeun-Jun Chung ◽

Minho Lee

Keyword(s):

Gene Expression ◽

Mrna Expression ◽

Cell Lines ◽

Cancer Cell ◽

Prediction Models ◽

Cancer Cell Lines ◽

Support Vector ◽

Rna Expression ◽

Expression Levels ◽

Protein Levels

Background: Due to the ease of quantifying mRNA expression in comparison with that of protein abundances, many studies have utilized it to infer protein product quantification. However, the mRNA expression values for a gene and its protein products are not known to have a strong relationship, because of the complex mechanisms required to regulate the amounts of protein levels, from translation to post-translational modifications. Methods: We have developed, in this study, models to predict protein levels from mRNA expression levels using the transcriptome and reverse phase protein arrays (RPPA)-based on protein levels in pan-cancer cell lines. When predicting the abundance of a protein expression, in addition to using RNA expression of the corresponding gene, we also used RNA expression levels of a particular set of other genes. By applying support vector regression, we have identified a 47-gene expression panel that contributes to the improved performance of the prediction, and its optimal subsets specific to each protein species. Result and Conclusion: Eventually, our final prediction models doubled the number of predictable protein expressions (r > 0.7). Due to the weaknesses of RPPA, our model had some limitations, however, we expect that these prediction models and the panel can be widely used in the future to infer protein abundances.

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