MEC1 Chronic Lymphocytic Leukemia Cell Research Articles

Translational development of a novel BAFF-R CAR-T therapy targeting B-cell lymphoid malignancies.

Several CD19-targeting CAR-T cells are used to treat leukemias and lymphomas; however, relapsed and/or refractory (R/R) disease is still observed in a significant number of patients. Additionally, the success of CD19-CAR-T cell therapies is not uniform across hematological malignancies, particularly in chronic lymphocytic leukemia (CLL). In this study, we present the development of a novel CAR-T cell therapy targeting B-cell activating factor receptor (BAFF-R), a key regulator of B-cell proliferation and maturation. A new monoclonal antibody against BAFF-R was generated from a hybridoma clone and used to create a novel MC10029 CAR construct. Through a series of in vitro and in vivo models using the Nalm-6 cell line for leukemia and the Z138 cell line for lymphoma, we demonstrated the antigen-specific cytotoxicity of MC10029 CAR-T cells against tumor cells. Additionally, MC10029 CAR-T cells exhibited potent antitumor effects against CD19 knockout tumor cells, mimicking CD19-negative R/R disease. MC10029 CAR-T cells were specifically targeted to CLL, in which BAFF-R is nearly always expressed. The cytotoxicity of MC10029 CAR-T cells was first shown in the MEC-1 CLL cell line, before we turned our efforts to subject-derived samples. Using healthy donor-engineered MC10029 CAR-T cells against enriched primary tumor cells, followed by subject-derived MC10029 CAR-T cells against autologous tumor cells, we showed the efficacy of MC10029 CAR-T cells against CLL subject samples. With these robust data, we have advanced to the production of MC10029 CAR-T cells, using GMP lentivirus, and obtained an IND approval in preparation for a Phase 1 clinical trial.

Patient-derived Siglec-6-targeting antibodies engineered for T-cell recruitment have potential therapeutic utility in chronic lymphocytic leukemia

BackgroundDespite numerous therapeutic options, safe and curative therapy is unavailable for most patients with chronic lymphocytic leukemia (CLL). A drawback of current therapies such as the anti-CD20 monoclonal antibody (mAb)...

The TAp63/BCL2 axis represents a novel mechanism of clinical aggressiveness in chronic lymphocytic leukemia

AbstractThe TA-isoform of the p63 transcription factor (TAp63) has been reported to contribute to clinical aggressiveness in chronic lymphocytic leukemia (CLL) in a hitherto elusive way. Here, we sought to further understand and define the role of TAp63 in the pathophysiology of CLL. First, we found that elevated TAp63 expression levels are linked with adverse clinical outcomes, including disease relapse and shorter time-to-first treatment and overall survival. Next, prompted by the fact that TAp63 participates in an NF-κB/TAp63/BCL2 antiapoptotic axis in activated mature, normal B cells, we explored molecular links between TAp63 and BCL2 also in CLL. We documented a strong correlation at both the protein and the messenger RNA (mRNA) levels, alluding to the potential prosurvival role of TAp63. This claim was supported by inducible downregulation of TAp63 expression in the MEC1 CLL cell line using clustered regularly interspaced short palindromic repeats (CRISPR) system, which resulted in downregulation of BCL2 expression. Next, using chromatin immunoprecipitation (ChIP) sequencing, we examined whether BCL2 might constitute a transcriptional target of TAp63 and identified a significant binding profile of TAp63 in the BCL2 gene locus, across a genomic region previously characterized as a super enhancer in CLL. Moreover, we identified high-confidence TAp63 binding regions in genes mainly implicated in immune response and DNA-damage procedures. Finally, we found that upregulated TAp63 expression levels render CLL cells less responsive to apoptosis induction with the BCL2 inhibitor venetoclax. On these grounds, TAp63 appears to act as a positive modulator of BCL2, hence contributing to the antiapoptotic phenotype that underlies clinical aggressiveness and treatment resistance in CLL.

Defining the Biochemical Role of Sialic Acid-Binding Immunoglobulin-like Lectin-6 in Adhesion and Migration in Chronic Lymphocytic Leukemia

Introduction: Sialic acid-binding immunoglobulin-like lectins (Siglec) are a group of lectins that regulate innate and adaptive immune function via glycan recognition. We and others have shown overexpression of Siglec-6, a member of Siglec family, on B cells from patients with chronic lymphocytic leukemia (CLL) compared to normal donor derived B cells. While placental expression of Siglec-6 has been shown to regulate invasion of trophoblast cells by binding to glycodelin, the biochemical role of Siglec-6 in CLL patients is not known. We describe here for the first time the functional relevance of Siglec-6 and its ligand sialyl Tn (sTn) in cell adhesion and migration in CLL. Biochemical mechanisms of Siglec-6 mediated cell adhesion and migration through DOCK8 dependent activation of Cdc42 associated with actin polymerization in CLL cells are presented. Further, the physiological relevance of Siglec-6/ DOCK8 axis in CLL cell adhesion and migration is validated using primary CLL patient samples, and genetically engineered loss of function Siglec-6 and DOCK8 mutant MEC1 CLL cell line. These studies thus elucidate the biological role of Siglec-6 in malignant CLL B cells and demonstrate therapeutic opportunities targeting Siglec-6 in CLL.Methods: Flow cytometry was used to analyze surface expression of Siglec-6 and sTn in CLL patients and normal donors. Transwell migration assay was used to assess in-vitro migratory role of Siglec-6. Mass spectrometry analysis was performed to identify Siglec-6 interacting proteins. CRISPR/Cas9 technique was used to generate knock-out (KO) cell lines for mechanistic studies. Phalloidin staining followed by confocal imaging was used to examine actin polymerization. Cdc42 activation was evaluated using a commercial kit which uses specialized PAK1-PBD agarose beads to pull down GTP-bound Cdc42. To study the in-vivo migratory role of Siglec-6, MEC1 CLL cell line or primary CLL cells were blocked with an isotype antibody or Siglec-6 targeted antibody and injected into the tail vein of NSG immunocompromised mice. 24 hrs later, mice were euthanized and spleens and BM were processed followed by flow cytometry analysis to determine the number of human CD45+ cells that have migrated.Results: We confirmed Siglec-6 overexpression on B cells from CLL patients when compared to B cells from normal donors. Interestingly, we also found expression of sTn on bone marrow stromal cells (BMSCs) derived from CLL patients but not healthy donors. Compared to Siglec-6 + CLL cells, Siglec-6 - CLL cells exhibited significant reduction in adhesion to (~50%) and migration towards (~50%) media containing sTn or sTn + CLL-BMSCs in cell adhesion and trans-well migration assays. Importantly, a Siglec-6 targeted antibody inhibited homing of Siglec-6 + MEC1 cells and primary CLL cells to the spleen and bone marrow in NSG mice (~35%). Mass spectrometry and co-immunoprecipitation analysis in MEC1 cells revealed interaction of Siglec-6 with DOCK8, a guanine nucleotide exchange factor. Stimulation of Siglec-6 + MEC1 cells with sTn resulted in Cdc42 activation and WASP protein recruitment, which are both downstream targets of DOCK8 involved in cell migration. Further, sTn also promoted actin polymerization, an effect that was compromised in Siglec-6 or DOCK8 KO MEC1 cells. Additionally, cell fractionation experiments revealed that Siglec-6 + MEC1 cells had higher levels of DOCK8 at the cell membrane when compared to MEC1 Siglec-6 KO cells, indicating that Siglec-6 may be responsible for tethering DOCK8 to the cell membrane.Conclusions: We have for the first time shown Siglec-6 dependent recruitment of DOCK8 leading to migration and adhesion of B-CLL cells. Siglec-6 signals via DOCK8 to mediate sTn ligand dependent actin polymerization. We have also shown that sTn promotes Cdc42 activation and WASP protein recruitment which are both essential for actin polymerization. Moreover, all these effects were prevented by CRISPR/Cas9 mediated knock out of Siglec-6 or DOCK8 in MEC1 CLL cell line. Thus, Siglec-6 represents a CLL-specific target that opens up new therapeutic avenues to target only malignant B-CLL cells. Ongoing studies are focused on determining molecular mechanisms of Siglec-6 mediated regulation of actin polymerization and CLL-BMSC interactions.[This work was supported by NIH-R21 Grant and Pelotonia Idea grants. JN is a recipient of Pelotonia Graduate Fellowship] DisclosuresByrd: Newave: Membership on an entity's Board of Directors or advisory committees; Vincerx Pharmaceuticals: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Novartis, Trillium, Astellas, AstraZeneca, Pharmacyclics, Syndax: Consultancy, Honoraria.

RPS15 mutations rewire RNA translation in chronic lymphocytic leukemia

AbstractRecent studies of chronic lymphocytic leukemia (CLL) have reported recurrent mutations in the RPS15 gene, which encodes the ribosomal protein S15 (RPS15), a component of the 40S ribosomal subunit. Despite some evidence about the role of mutant RPS15 (mostly obtained from the analysis of cell lines), the precise impact of RPS15 mutations on the translational program in primary CLL cells remains largely unexplored. Here, using RNA sequencing and ribosome profiling, a technique that involves measuring translational efficiency, we sought to obtain global insight into changes in translation induced by RPS15 mutations in CLL cells. To this end, we evaluated primary CLL cells from patients with wild-type or mutant RPS15 as well as MEC1 CLL cells transfected with mutant or wild-type RPS15. Our data indicate that RPS15 mutations rewire the translation program of primary CLL cells by reducing their translational efficiency, an effect not seen in MEC1 cells. In detail, RPS15 mutant primary CLL cells displayed altered translation efficiency of other ribosomal proteins and regulatory elements that affect key cell processes, such as the translational machinery and immune signaling, as well as genes known to be implicated in CLL, hence highlighting a relevant role for RPS15 in the natural history of CLL.

DAP Kinase-Related Apoptosis-Inducing Protein Kinase 2 (DRAK2) Is a Key Regulator and Molecular Marker in Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the Western World and it is characterized by a marked degree of clinical heterogeneity. An impaired balance between pro- and anti-apoptotic stimuli determines chemorefractoriness and outcome. The low proliferation rate of CLL cells indicates that one of the primary mechanisms involved in disease development may be an apoptotic failure. Here, we study the clinical and functional significance of DRAK2, a novel stress response kinase that plays a critical role in apoptosis, T-cell biology, and B-cell activation in CLL. We have analyzed CLL patient samples and showed that low expression levels of DRAK2 were significantly associated with unfavorable outcome in our CLL cohort. DRAK2 expression levels showed a positive correlation with the expression of DAPK1, and TGFBR1. Consistent with clinical data, the downregulation of DRAK2 in MEC-1 CLL cells strongly increased cell viability and proliferation. Further, our transcriptome data from MEC-1 cells highlighted MAPK, NF-κB, and Akt and as critical signaling hubs upon DRAK2 knockdown. Taken together, our results indicate DRAK2 as a novel marker of CLL survival that plays key regulatory roles in CLL prognosis.

CG-806, a First-in-Class Pan-FLT3/Pan-BTK Inhibitor, Exhibits Broad Signaling Inhibition in Chronic Lymphocytic Leukemia Cells

CG-806, a First-in-Class Pan-FLT3/Pan-BTK Inhibitor, Exhibits Broad Signaling Inhibition in Chronic Lymphocytic Leukemia Cells

Abstract 4493: SF3B1 mutation promotes c-Myc protein stability through aberrant splicing and downregulation of PP2A B56α subunit in chronic lymphocytic leukemia

Abstract SF3B1 mutation, which occurs in 10-20% of chronic lymphocytic leukemia (CLL) patients, is associated with faster disease progression, shorter overall survival and fludarabine resistance. Mutant SF3B1 utilizes cryptic 3′ splice sites to generate aberrantly spliced mRNAs, half of which may undergo non-sense mediated mRNA decay (NMD), leading to downregulation of the affected genes. To identify potential tumor suppressor genes that are aberrantly spliced and downregulated by mutant SF3B1, we performed RNA-sequencing analysis of SF3B1-mutated primary CLL cells in the presence or absence of cycloheximide (CHX), a translation inhibitor known to inhibit NMD. Our analysis identified PPP2R5A, encoding one of the regulatory subunits of protein phosphatase-2A (PP2A B56α), as one of the key genes that were most consistently affected by an aberrantly spliced junction, and significantly downregulated (>2 fold) in CLL patient samples with various SF3B1 hotspot mutations. Splicing analyses by RT-PCR and Sanger sequencing confirmed that a 13-nucleotides sequence was added before the 5th exon of PPP2R5A via alternative splicing in CLL patients with the SF3B1 mutation. Due to this 13-nucleotides addition, three consecutive premature stop codons were created by frameshift at a position more than 55 bases upstream of an exon-exon junction, which is a canonical feature of the mRNAs degraded through NMD. The down-regulation of PPP2R5A was confirmed by quantitative PCR (p<0.05) and immunoblot at both mRNA and protein levels in CLL patients with SF3B1 mutation as compared to CLL patients without SF3B1 mutation and normal CD19+ B cell samples. Furthermore, overexpression of mutant SF3B1 in K562 and HEK 293 cells resulted in the same aberrant splicing pattern observed in CLL patients with SF3B1 mutation and downregulation of PPP2R5A (p<0.05). Treatment with CHX resulted in an increased abundance of alternatively spliced PPP2R5A transcript, suggesting that the aberrant PPP2R5A transcript was degraded by NMD. PP2A, when incorporated with PPP2R5A as the regulatory subunit, dephosphorylates c-Myc at S62, and results in the degradation of c-Myc. Here, we demonstrated that downregulation of PPP2R5A by mutant SF3B1 led to upregulation of c-Myc through promoting c-Myc protein stability in SF3B1K700E overexpressing cells when compared to SF3B1wildtype overexpressing cells. Knockdown PPP2R5A in MEC1 CLL cells by siRNA also significantly upregulated c-Myc at the protein level and upregulated mRNA expression of c-Myc downstream genes NCL and ODC1. Overall, our results demonstrate that SF3B1 mutation causes aberrant splicing and downregulation of tumor suppressor protein PP2A B56α in vitro and in vivo, leading to upregulation of c-Myc. Citation Format: Qimei Han, Libin Deng, Shuo Tu, Lirong Pei, Jeong-Hyeon Choi, Victor Jin, Huidong Shi. SF3B1 mutation promotes c-Myc protein stability through aberrant splicing and downregulation of PP2A B56α subunit in chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4493.

Abstract 5230: Modulation of SF3B1 causes global intron retention and downregulation of the B-cell receptor pathway in chronic lymphocytic leukemia

Abstract Splicing factor SF3B1 is frequently mutated in chronic lymphocytic leukemia (CLL) patients and has been suggested as a potential therapeutic target. In this study, we demonstrated that SF3B1 modulator sudemycin D6 (SD6) effectively inhibits cell growth and induces apoptosis in CLL cells. RNA sequencing analysis revealed significant increases in global intron retention in SD6-treated CLL cells. Pathway analysis of the genes associated with increased intron retention suggested that B-cell receptor (BCR) and PI3K signaling pathways were among the most important pathways being affected by SD6. The increases in intron retention were inversely correlated with decreases in mRNA and protein levels of the affected BCR/PI3K pathway molecules including BLNK, BTK, AKT, PLCγ2, and PI3Kδ. SD6 treatment also induced a time-dependent exon-skipping event in MCL-1 mRNA and resulted in significant down-regulation of another anti-apoptotic gene TRAF1, thus collectively contributing to the SD6-induced apoptosis. Furthermore, SD6 treatment can overcome the pro-survival and pro-growth signals and synergize with established CLL therapies ibrutinib, idelalisib, and venetoclax to induce apoptosis in primary CLL cells co-cultured with bone marrow stromal cells and T-cell-derived cytokines. Finally, in vivo treatment with SD6 at 10mg/kg/day for 7 days significantly inhibited the growth of xenograft tumors that were established by subcutaneous inoculation of 5×106 MEC1 CLL cells into NOD mice. Collectively, these results provide a strong rationale for the future clinical development of spliceosome modulators and potential combination therapies for the treatment of CLL. Citation Format: Qimei Han, Jianbo Wang, Austin Y. Shull, Fang Shi, Libin Deng, Jeong-Hyeon Choi, Eun-Jeong Park, Shuo Tu, Lirong Pei, Farrukh T. Awan, Roni Bollag, Locke J. Bryan, Hong-bo Xin, Chandraiah Lagisetti, Thomas R. Webb, Victor Jin, Huidong Shi. Modulation of SF3B1 causes global intron retention and downregulation of the B-cell receptor pathway in chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5230.

Activating MAPK Pathway Mutations Mediate Primary Resistance to PI3K Inhibitors in Chronic Lymphocytic Leukemia (CLL)

Resistance to PI3K inhibitors including idelalisib remains poorly understood. The present study was designed to identify mechanisms underlying PI3K inhibitor resistance in CLL through WES, RNAseq and biochemical confirmation. We performed WES on matched tumor and germline samples from 28 relapsed CLL patients treated with PI3K inhibitors (idelalisib, 79% (22/28), pilaralisib 10.7% (3/28), voxtalisib 10.7% (3/28)). The median number of prior therapies was 4.5 and the adverse cytogenetic aberrations del(17p) and del(11q) were seen in 21% and 32% respectively. A median of two longitudinal tumor samples were sequenced per patient (Nsamples= 68). An average of 23 ± 12 (range = 4-58) non-silent somatic mutations were identified in the initial sample. The non-silent mutation rate was 0.64 ± 0.32 per MB (range = 0.11-1.61) and typical of CLL. Significantly mutated genes (based on MutSig2CV) included SF3B1 36%, TP53 21%, DDX3X 14% and BRAF 11%. The cohort was sub-divided into responders (R) (Ncases= 18, Nsamples= 47) and non-responders (NR) (Ncases= 10, Nsamples= 21). Among the 10 NR, 7 had been treated with idelalisib and 3 with voxtalisib. Comparison of the mutational landscape between R and NR showed that, notably, mutations in BRAF(N=5), MAP2K1(N=2), KRAS(N=2),XPO1(N=1), PLEKHA1(N=1), INPPL1(N=1) and NXF1(N=1) were exclusively found in NR. Moreover, MAPK pathway genes (KRAS, BRAF and MAP2K1) were the only recurrent somatic mutations present exclusively in NR. We inferred the cancer cell fraction (ccf) for each somatic mutation using ABSOLUTE and found that the ccf values for MAP2K1 (p=0.01), XPO1 and PLEKHA1 mutations increased with time, indicating a positive selection pressure. Phylogic analysis of 6 R and 4 NR with at least 3 serial samples identified a median of 2 dynamic subclones (change of at least 10% between any two timepoints) and showed enrichment of the “Reactome_RAF_MAP_Kinase_Cascade” pathway in NR (p=3x10-4, q=0.04). In addition, Gene Set Enrichment Analysis comparing RNAseq samples drawn prior to or after PI3K therapy in five of the patients from our cohort (1 primary NR and 4 progressors after response) demonstrated activation of the “Reactome_Prolonged_ERK_Activation_Events” pathway at the later time-points (p=2.3x10-4, q= 0.03).Given these findings, we further investigated the role of the ERK/MAPK pathway in idelalisib resistance. Western blot analysis of ERK1/2 phosphorylation in ex vivo idelalisib treated PBMCs from an initially responding patient who acquired resistance later showed that idelalisib failed to inhibit ERK1/2 phosphorylation only at the resistant timepoint. This persistent ERK1/2 activation was inhibited by combination treatment with the MAP2K1 inhibitor CI-1040. We then profiled pAKT and pERK relative to total, by western blot on ex vivo idelalisib-treated samples (5 R, 5 NR). Idelalisib treatment ex vivo inhibited AKT phosphorylation in both R and NR, but no reduction in ERK phosphorylation was seen in NR, in contrast to R.Based on these findings we hypothesized that increased or persistent ERK activity confers resistance to PI3K inhibitors and tested the impact of MAP2K1 mutations on ERK activity. Among NR, we identified two different mutations in MAP2K1, E203K and Q56P (2/10 NR vs 0/18 R), both previously described as activating in solid tumors. In a separate cohort of multiply relapsed CLL patients, we found 4 cases of MAP2K1 F53L mutations, also previously reported in solid tumors. We studied the functional significance of these MAP2K1 mutations by generating stable MEC1 CLL cell lines expressing HA tagged E203K, F53L or Q56P MAP2K1 mutants. We found that all mutant cell lines had elevated basal ERK1/2 phosphorylation (E203K p<0.001; F53L p<0.001; Q56P p<0.001). Moreover, idelalisib did not inhibit IgM-induced ERK1/2 phosphorylation in the mutants when compared to the controls expressing wild type MAP2K1 (E203K p<0.001; F53L p<0.001; Q56P p<0.001). Preliminary results using the ERK1/2 inhibitor SCH772984 showed that the combination of SCH772984 along with idelalisib effectively decreased ERK1/2 phosphorylation in the mutant cell lines compared to idelalisib alone.Taken together, these data implicate the ERK pathway in idelalisib resistance and suggest that ERK1/2 inhibitors either combined with idelalisib at therapy start or added to idelalisib at early progression might sensitize patients to PI3K delta therapy. DisclosuresBrown:Acerta / Astra-Zeneca: Membership on an entity's Board of Directors or advisory committees; Invectys: Membership on an entity's Board of Directors or advisory committees; Sunesis: Consultancy; TG Therapeutics: Consultancy; Morphosys: Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Consultancy; Gilead: Consultancy, Research Funding; Celgene: Consultancy; Genentech: Consultancy; Pharmacyclics: Consultancy; Abbvie: Consultancy; Verastem: Consultancy, Research Funding; Beigene: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy; Sun Pharmaceutical Industries: Research Funding; Loxo: Consultancy; Boehringer: Consultancy.

RPS15 mutations Repress mRNA Translation in Chronic Lymphocytic Leukemia Cells

We and others recently reported mutations within the RPS15 gene, encoding a component of the 40S ribosomal subunit, in clinically aggressive chronic lymphocytic leukemia (CLL). RPS15 mutations resided within an evolutionary conserved region, alluding to an oncogenic rather than a tumor-suppressor role. Our pilot functional analysis revealed that, similar to other ribosomal proteins (RPs), RPS15 also binds MDM2 and may impact on the p53 response. Here, we performed ribosome profiling in order to gain global insight into changes in translation induced by RPS15 mutations in CLL cells. This technique involves measuring translational efficiency (TE), by comparing the levels of ribosome-associated mRNA footprints against the total mRNA for each gene. For 6 CLL cases bearing mutant (mut, n=3) or wildtype (wt, n=3) RPS15, we obtained both ribosome-protected footprints (RPFs) and matching mRNA sequencing data. In parallel, we created stable MEC1 CLL cell lines expressing an additional copy of wt or mut RPS15 (131S) by lentiviral transduction; validation of the transgene expression was performed by Sanger sequencing of amplified cDNAs. Ribosome footprinting and subsequent library preparation of RPFs and total mRNA for all samples was performed with the Illumina Truseq Ribo Profile Kit and all libraries were sequenced on a NextSeq500 instrument. Reads were aligned to the human hg19 genome using Bowtie2. SystemPipeR was used to determine the percentage of reads mapping to 5‘ UTRs, CDS, and 3‘ UTRs and triplet periodicity was assessed using RibORF. The RPFs were of high quality, as assessed by expected RPF size (28-30nt), CDS enrichment, and triplet periodicity.To determine differentially expressed genes between RPS15-mut vs RPS15-wt cases we used DESeq2 while, for differentially translated genes we used Xtail. Changes in transcription and translation between PRS15-wt vs RPS15-mut cases showed limited overlap in both primary CLL cells and cell lines (12.8% and 12.9%, respectively), indicating the potential of ribosome profiling to reveal additional information compared to RNA sequencing alone. In primary CLL cells, 474 genes showed differences only at the transcription level (log2FC mRNA>I1I, p<0.05), while 742 genes were modulated only at the translation level (log2FC RPF>I1I, p<0.05). We identified 322 genes with differential TE (log2FC TE<I1I, p<0.05) between PRS15-wt vs RPS15-mut CLL cases; 262/322 (81%) showed reduced TE in RPS15-mut versus RPS15-wt cases. Similar analysis for the stable MEC1 cell lines revealed 749 genes displaying differences only at the transcription level (log2FC mRNA>I1I), while 1859 genes were regulated only at the translation level (log2FC RPF>I1I). Overall, 771 genes displayed differential TE (log2FC TE<I1I, adjusted p<0.1) between PRS15-wt vs RPS15-mut MEC1 cell lines; 48% of the genes showed reduced TE in mut vs wt cell lines and the remaining 52% augmented TE. The slightly different results compared to those obtained from primary CLL cells, may be attributed to the following reasons: (i) MEC1 cells are TP53-aberrant; (ii) the PRS15-wt cell line overexpresses the RPS15 gene compared to primary CLL cells; and,( iii) the RPS15-mut cell line expresses both the wt and mut RPS15 mRNAs (22% of the mapped reads correspond to the mut RPS15 and 78% to the wt gene, respectively). Gene ontology analysis (Enrichr) of the genes showing differential TE, revealed that in both primary CLL cells and MEC1 cell lines a large fraction of the deregulated transcripts is implicated in RNA binding processes (adj-p=0.0001; adj-p=1.98X10^-13, respectively) which are known to induce translational repression. Interestingly, in primary CLL cells, amongst genes with reduced TE we identified genes implicated in tRNA biosynthesis, protein processing in the endoplasmatic reticulum and the Hippo signaling pathway (p<0.01). Additionally, enrichment analysis revealed that a proportion of genes with reduced TE were targets of the MYC transcription factor (adj-p=0.0005). RP genes, despite unchanged mRNA levels, showed changes in RPF levels and differential TE, suggesting that RPs are also deregulated at the translational level. In conclusion, we show that RPS15 mutations rewire the translation program of CLL cells by reducing the TE of critical molecules, including translation initiation factors and other regulatory elements. DisclosuresHadzidimitriou:Abbvie: Research Funding; Gilead: Research Funding; Janssen: Honoraria, Research Funding. Stamatopoulos:Janssen: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; Gilead: Honoraria, Research Funding.

The Transcription Factor TAp63 Exerts Pro-Survival Effects in Chronic Lymphocytic Leukemia Acting through the BCL2 Pathway

Recent evidence indicates that TAp63, the prevalent isoform of TP63 in Chronic Lymphocytic Leukemia (CLL), is implicated in disease pathogenesis. In CLL, TAp63 expression, modulated by both immune signaling and epigenetic modifications, promotes leukemic cell survival and homing to the bone marrow. In activated normal B cells, the TAp63 transcription factor binds the BCL2 gene, participating in an anti-apoptotic pathway (axis NF-κB/TAp63/BCL2) augmenting cell survival. In this study, we investigated the expression of TAp63 in a large cohort of CLL cases and its potential fluctuation during disease progression. Additionally, in order to further understand the pro-survival role of TAp63 in CLL, we interrogated at the molecular level the interplay betweenΤAp63 and BCL2.Initially, using RT-qPCR we quantified TAp63 mRNA expression in 166 CLL patients, consisting of 89 with unmutated IGHV genes (U-CLL) and 77 with mutated IGHV genes (M-CLL), prior to administration of treatment. Significantly higher TAp63 mRNA levels were observed in U-CLL vs M-CLL (FD=13.83, p<0.0001). However, outliers were identified in both subgroups, prompting us to re-classify all cases into TAp63high and TAp63low subgroups using ROC curve and Youden index statistical procedures. TAp63high patients displayed significantly shorter time-to-first-treatment (TTFT) (TAp63highmedian TTFT: 1.58 years; TAp63lowmedian TTFT: 4.07 years; p=0.03) and shorter overall survival (OS) (TAp63highmedian OS: 7.825 years; TAp63lowmedian OS: not yet reached; p=0.046). Next, we analyzed TAp63 mRNA expression in longitudinal samples of 25 U-CLL cases treated with either FCR or rituximab-chlorambucil. In each case, samples were collected at three ‘landmarks’; diagnosis, first progressionand first relapse. Expression analysis by RT-qPCR showed that TAp63 levels significantly increased at disease relapse compared to diagnosis (FD=3.47, p=0.02).We subsequently investigated links between TAp63 and BCL2 by measuring BCL2 mRNA levels in 56 U-CLL cases from the present cohort and found statistically significant correlation with the corresponding TAp63 mRNA levels (spearman rho=0.31, p=0.01). To validate this observation, we undertook functional studies in the MEC1 CLL cell line. Considering that MEC1 cells express high TAp63 mRNA levels, we generated a stable MEC1 cell line to inducibly downregulate TAp63, using CRISPR/dCas9-KRAB upon treatment with doxycycline (Dox). We used 2 different guide RNAs (sgRNAs; sgRNA1, sgRNA2) targeting 2 distinct regions of the endogenous TAp63 promoter. After 5 days of induction, the expression levels of both TAp63 and BCL2 were quantified by one step RT-qPCR in Tet-on-dCas9-KRAB-sgRNA-TAp63 MEC1 cells. Inducible downregulation of TAp63 expression (gRNA1: FD=1.7, gRNA2: FD=1.53) resulted in downregulation of BCL2 expression (gRNA1: FD=1.34, gRNA2: FD=1.12) with strong correlation (rho=0.97, p<0.0001) between TP63 and BCL2 mRNA levels. Furthermore, we also observed correlation between TAp63 and BCL2 protein expression in primary cells of one representative TP63high CLL case (rho=0.94, p=0.01), in which TAp63 was silenced by RNA interference (RNAi) with 3 different siRNAs. Prompted by these results, we additionally assessed ex vivo the effect of the BCL2 inhibitor Venetoclax in primary CLL cells of both TAp63high (n=8) and TAp63low (n=6) cases. Cell viability was measured by flow cytometry at 24 and 48 hours after treatment. TAp63high cases were more resistant to treatment with Venetoclax as they showed no statistically significant reduction in cell viability compared to the respective (DMSO-treated) controls, in contrast to TAp63low cases (24h: FD=3.63, p=0.004; 48h: FD=7.17, p=0.005).In conclusion, we provide evidence suggesting that up-regulated TAp63 expression represents a novel resistance mechanism to chemoimmunotherapy in CLL. The pro-survival role of TAp63 is supported by its strong association with BCL2. Indeed, based on the present findings, TAp63 appears to act as a positive modulator of BCL2 in CLL cells, rendering them less responsive to apoptosis induction with the BCL2 inhibitor Venetoclax. DisclosuresHadzidimitriou:Abbvie: Research Funding; Gilead: Research Funding; Janssen: Honoraria, Research Funding. Stamatopoulos:Abbvie: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Janssen: Honoraria, Research Funding.

GLP overexpression is associated with poor prognosis in Chronic Lymphocytic Leukemia and its inhibition induces leukemic cell death.

Background Heterodimeric methyltransferases GLP (EHMT1/KMT1D) and G9a (EHMT2/KMT1C) are two closely related enzymes that promote the monomethylation and dimethylation of histone H3 lysine 9. Dysregulation of their activity has been implicated in several types of human cancer. Patients and methods Here, in order to investigate whether GLP/G9a exerts any impact on Chronic Lymphocytic Leukemia (CLL), GLP/G9a expression levels were assessed in a cohort of 50 patients and the effects of their inhibition were verified for the viability of CLL cells. Also, qRT-PCR was used to investigate the transcriptional levels of GLP/G9a in CLL patients. In addition, patient samples were classified according to ZAP-70 protein expression by flow cytometry and according to karyotype integrity by cytogenetics analysis. Finally, a selective small molecule inhibitor for GLP/G9a was used to ascertain whether these methyltransferases influenced the viability of MEC-1 CLL cell lineage. Results mRNA analysis revealed that CLL samples had higher levels of GLP, but not G9a, when compared to non-leukemic controls. Interestingly, patients with unfavorable cytogenetics showed higher expression levels of GLP compared to patients with favorable karyotypes. More importantly, GLP/G9a inhibition markedly induced cell death in CLL cells. Conclusion Taken together, these results indicate that GLP is associated with a worse prognosis in CLL, and that the inhibition of GLP/G9a influences CLL cell viability. Altogether, the present data demonstrate that these methyltransferases can be potential markers for disease progression, as well as a promising epigenetic target for CLL treatment and the prevention of disease evolution.

Inhibition of chronic lymphocytic leukemia progression by full-length chromogranin A and its N-terminal fragment in mouse models

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of leukemic B cells in peripheral blood, bone marrow (BM) and lymphoid tissues, and by their recirculation between these compartments. We observed that circulating chromogranin A (CgA) and its N-terminal fragment (called vasostatin-1, CgA1-76), two neuroendocrine secretory polypeptides that enhance the endothelial barrier function, are present in variable amounts in the blood of CLL patients. Studies in animal models showed that daily administration of full-length human CgA1-439 (0.3 μg, i.v., or 1.5 μg/mouse, i.p.) can reduce the BM/blood ratio of leukemic cells in Eμ-TCL1 mice, a transgenic model, and decrease BM, lung and kidney infiltration in Rag2−/−γc−/− mice engrafted with human MEC1 CLL cells, a xenograft model. This treatment also reduced the loss of body weight and improved animal motility. In vitro, CgA enhanced the endothelial barrier integrity and the trans-endothelial migration of MEC1 cells, with a bimodal dose-response curve. Vasostatin-1, but not a larger fragment consisting of N-terminal and central regions of CgA (CgA1-373), inhibited CLL progression in the xenograft model, suggesting that the C-terminal region is crucial for CgA activity and that the N-terminal domain contains a site that is activated by proteolytic cleavage. These findings suggest that circulating full-length CgA and its fragments may contribute to regulate leukemic cell trafficking and reduce tissue infiltration in CLL.

Bruton's tyrosine kinase inhibitor restrains Wntsignaling in chronic lymphocytic leukemia.

The B-cell receptor (BCR) signaling pathway serves an important role in the pathogenesis of chronic lymphocytic leukemia (CLL), and has been identified as a novel and effective therapeutic target of CLL, with particular focus its kinase factor, BTK. Previous studies have focused on combining the BTK inhibitor with additional chemotherapeutic agents to improve the prognosis of patients with CLL. Further investigation into the mechanism of the BTK inhibitor would promote an understanding of the pathogenesis of CLL. The current study investigated the association between ibrutinib and the Wnt signaling pathway, additionally focussing upon one of its regulators, metadherin (MTDH), which has been identified to be overexpressed in CLL and is considered a promoter of the Wnt pathway. The experiments in the current study were performed in the MEC-1 CLL cell line. Results indicated that MTDH, β-catenin and lymphoid-enhancing factor-1 were inhibited subsequent to ibrutinib treatment. The results indicate that in CLL, ibrutinib is likely to possess an inhibitory role in Wnt signaling.

Mirna-21 Targets Sprouty2 to Activate SYK Mediated BCR and MAPK-Erk Signaling in Chronic Lymphocytic Leukemia

Abstract INTRODUCTION: Chronic Lymphocytic Leukemia (CLL) represents the most common adult leukemia in the western world. Several reports have demonstrated that the constitutively activated MAPK-Erk signaling pathways promote CLL cell proliferation and survival. However, the precise molecular mechanisms that lead to deregulated MAPK signaling in CLL initiation and progression are not fully understood. We have previously reported that Sprouty2 (Spry2) is a negative regulator of BCR and MAPK-Erk signaling in poor prognosis CLL (American Society of Hematology, 56th Annual Meeting, San Francisco, CA, 2014). Here in follow up studies, we set to further determine molecular basis deregulation of Spry2 in patients with poor prognosis CLL. Spry2 is either epigenetically silenced or targeted by Microrna-21 (miR21) in several malignancies. MiR-21 plays a role of oncomir in CLL, which is significantly upregulated in CLL patients with poor prognosis specifically with Del 17p, High 38 and Zap70 expression. Notably, we observed a decrease in Spry2 expression in CLL cells expressing high levels of miR-21. Also, it is already reported that miR21 is regulated by STAT3 transcription factor in CLL patients with poor prognosis in refractory phase. However the precise molecular mechanism by which the miR21 leads to leukemic progression in poor prognosis CLL is not known. Here we demonstrate the molecular mechanism of miR21 to activate Syk mediated BCR and MAPK-Erk signaling by targeting Spry2 in CLL cells from poor prognosis patients. METHODOLOGY: We isolated CLL cells from peripheral blood of newly diagnosed high CD38 poor prognosis patients. For molecular analysis, in addition to primary CLL cells we use Mec-1 CLL cell line. To measure the levels of miR21 we used Taqman assay qPCR from Invitrogen. To decrease miR21 levels we used miR21 inhibitor (Invitrogen) and STAT3 inhibitor (SantaCruz). To overexpress miR21 we used pcDNAmir-21 construct from Addgene. Activation of BCR and MAPK-Erk signaling pathway was measured by levels of p-Erk and p-Syk. Expression of Spry2, Erk and Syk was measured by western blot analysis. RESULTS: To study if miR21 targets Spry2 in CLL cells, we first overexpressed miR21 in Mec-1 cells. We observed a significant decrease in Spry2 expression in Mec-1 cells expressing high levels of miR-21 compared to empty vector control. In our previous studies we observed Spry2 acts as a negative regulator of BCR and MAPK-Erk signaling. Therefore, we next studied the activity of Spry2 targeting signaling pathways in miR21-overexpressing CLL cells. We observed elevated levels of p-Erk and p-Syk in CLL cells overexpressing miR21. For control we used pcDNA-empty vector. Whereas upon miR21 knockdown in CLL cells resulted in stabilization of Spry2 expression and decrease in BCR and MAPK-Erk signaling. Also, targeting miR21 by STAT3 inhibitor leads to induction of spontaneous apoptosis in CLL cells. We next tested the effect of STAT3 inhibitor on primary CLL cells from 13 different patients. We measured the viability of CLL cells using MTT assay, we observed higher efficacy of STAT3 inhibitor over Syk and BRAF inhibitors on CLL cells. CONCLUSION: We conclude that Spry2 is a target of miR21 in CLL cells from poor prognosis patients. MiR21 targets Spry2 to activate Syk mediated BCR and MAPK signaling in CLL patients. Also, STAT3 can be used as therapeutic target for poor prognosis CLL patients with high miR21 expression. Disclosures No relevant conflicts of interest to declare.

Human ROR1 Activates AKT and Accelerates Leukemia Cell Proliferation

AbstractAbstract 3872ROR1 is an orphan-receptor-tyrosine-kinase-like developmental surface-antigen that is expressed by embryonic cells, chronic lymphocytic leukemia (CLL) cells, and neoplastic cells of many other types of cancer, but not by virtually all normal adult tissues. ROR1 can serve as a receptor for Wnt5a and potentially complex with other surface receptors for growth/survival factors elaborated by the tumor microenvironment or expressed by the tumor itself. In prior studies, we and others found that silencing ROR1 could impair tumor-cell growth and/or survival in vitro and in vivo. Although the CLL cells of ≥96% of all examined patients (N= 800) expressed ROR1, we found that a B-cell line derived from CLL cells, namely MEC1 (Leuk Res 23:127, 1999), lacked expression of this protein. This provided us with an opportunity to assess how introduction of ROR1 could affect the biology of this CLL-cell line, which has relatively slow doubling-time of ≈40 hours. We transfected MEC1 cells with either an expression vector encoding human ROR1 or a control vector, and then selected for stable transfectants in selection medium. MEC1 cells transfected with the ROR1 vector expressed high-levels of ROR1, but not MEC1 cells transfected with the control vector. MEC1 cells made to express ROR1 had higher levels of phosphorylated and activated-AKT and activated cAMP response-element-binding (CREB) protein than non-transfected MEC1 cells or cells transfected with the control vector. Furthermore, MEC1 made to express ROR1 had significantly greater proportions of cells in S/G2/M phase than did control-transfected cells 16 hours after transfer from serum-free medium to complete growth medium, indicating that MEC1 cells made to express ROR1 had increased relative proportions of cells undergoing cell division. Consistent with this, we found that ROR1+ MEC1 cells had significantly shorter doubling times in culture than did comparably cultured parental MEC1 cells or MEC1 cells transfected with the control vector. This study indicates that expression of ROR1 in the MEC1 CLL cell line can activate AKT/CREB and accelerate leukemia cell growth, providing us with a model system with which to interrogate the function of ROR1 in vitro. Disclosures:No relevant conflicts of interest to declare.

5.26 Hsp90 Inhibitor Restores P53-Mutated MEC1 Chronic Lymphocytic Leukemia Cell Sensitivity to Fludarabine by Downregulating DNA Repair and Endoplasmic Reticulum Chaperone Proteins

5.26 Hsp90 Inhibitor Restores P53-Mutated MEC1 Chronic Lymphocytic Leukemia Cell Sensitivity to Fludarabine by Downregulating DNA Repair and Endoplasmic Reticulum Chaperone Proteins