Leukemia Samples Research Articles

Glucocorticoid Resistant Pediatric Acute Lymphoblastic Leukemia Samples Display Altered Splicing Profile and Vulnerability to Spliceosome Modulation.

Glucocorticoid (GC) resistance is a crucial determinant of inferior response to chemotherapy in pediatric acute lymphoblastic leukemia (ALL); however, molecular mechanisms underlying this phenomenon are poorly understood. Deregulated splicing is a common feature of many cancers, which impacts drug response and constitutes an attractive therapeutic target. Therefore, the aim of the current study was to characterize global splicing profiles associated with GC resistance and determine whether splicing modulation could serve as a novel therapeutic option for GC-resistant patients. To this end, 38 primary ALL samples were profiled using RNA-seq-based differential splicing analysis. The impact of splicing modulators was investigated in GC-resistant leukemia cell lines and primary leukemic specimens. Our findings revealed, for the first time, markedly distinct splicing landscapes in ALL samples of B-cell precursor (BCP)-ALL and T-ALL lineages. Differential splicing events associated with GC resistance were involved in RNA processing, a direct response to GCs, survival signaling, apoptosis, cell cycle regulation and energy metabolism. Furthermore, our analyses showed that GC-resistant ALL cell lines and primary samples are sensitive to splicing modulation, alone and in combination with GC. Together, these findings suggest that aberrant splicing is associated with GC resistance and splicing modulators deserve further interest as a novel treatment option for GC-resistant patients.

Electrochemical determination of L-arginine in leukemic blood samples based on a polyaniline-multiwalled carbon nanotube—magnetite nanocomposite film modified glassy carbon electrode

The present study reports an electrochemical biosensor for the rapid and selective detection of L-arginine in leukemic blood samples. The biosensor was fabricated using a nanocomposite (PANI/MWCNTs/Fe3O4) film modified glassy carbon electrode (GCE). The partially purified arginine deiminsae (ADI) enzyme from P. putida was immobilized on the modified GCE by cross linking with gluteraldehyde. The characterization and validation of the developed biosensor was performed by various electrochemical techniques including cyclic voltammetry (CV), differential pulse voltammetry (DPV) and chronoamperometry (CA). The limit of detection for biosensor was determined to be as low as 20 μM based on a signal-to-noise ratio equal to three while employing a detection period of 10 s. A linear response for L-arginine was observed across the concentration range from 100 to 1000 µM with an R2 value of 0.9987. The biosensor designed in the present study offers highly specific and sensitive analysis in normal and leukemic blood samples compared to previous reports in the literature. Therefore this device has excellent potential for nano-device fabrication for biomedical applications.

Curcumin augments therapeutic efficacy of TRAIL-based immunotoxins in leukemia.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) has been perceived as a promising anti-cancer agent because of its unique ability to kill cancer cells while sparing normal cells. However, translation of TRAIL to clinical studies was less successful as a large number of cancer cells acquire resistance to TRAIL-based monotherapies. An ideal strategy to overcome TRAIL resistance is to combine it with potential sensitizing agents. To investigate the TRAIL-sensitizing effect of curcumin in leukemia. The mechanism underlying TRAIL sensitization by curcumin was studied by flow cytometric analysis of TRAIL receptors in leukemic cell lines and patient samples, and immunoblot detection of TRAIL-apoptosis signaling proteins. Curcumin augments TRAIL-apoptotic signaling in leukemic cells by upregulating the expression of DR4 and DR5 along with suppression of cFLIP and anti-apoptotic proteins Mcl-1, Bcl-xl, and XIAP. Curcumin pre-treatment significantly (p < 0.01) enhanced the sensitivity of leukemic cell lines to TRAIL recombinant proteins. IL2-TRAIL peptide in the presence of curcumin induced potent apoptosis (p <0.001) as compared to TRAIL and IL2-TRAIL protein in leukemic cell lines with IC50 < 0.1 μΜ. Additionally, the combination of IL2-TRAIL peptide and curcumin showed significant cytotoxicity in patient peripheral blood mononuclear cells (PBMCs) with an efficacy of 90% in acute myeloid leukemia (AML), but 100% in acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL) and chronic myelomonocytic leukemia (CMML). Overall, our results suggest that curcumin potentiates TRAIL-induced apoptosis through modulation of death receptors and anti-apoptotic proteins which significantly enhances the therapeutic efficacy.

Identifying IGH disease clones for MRD monitoring in childhood B-cell acute lymphoblastic leukemia using RNA-Seq.

Identifying patient-specific clonal IGH/TCR junctional sequences is critical for minimal residual disease (MRD) monitoring. Conventionally these junctional sequences are identified using laborious Sanger sequencing of excised heteroduplex bands. We found that the IGH is highly expressed in our diagnostic B-cell acute lymphoblastic leukemia (B-ALL) samples using RNA-Seq. Therefore, we used RNA-Seq to identify IGH disease clone sequences in 258 childhood B-ALL samples for MRD monitoring. The amount of background IGH rearrangements uncovered by RNA-Seq followed the Zipf's law with IGH disease clones easily identified as outliers. Four hundred and ninety-seven IGH disease clones (median 2, range 0-7 clones/patient) are identified in 90.3% of patients. High hyperdiploid patients have the most IGH disease clones (median 3) while DUX4 subtype has the least (median 1) due to the rearrangements involving the IGH locus. In all, 90.8% of IGH disease clones found by Sanger sequencing are also identified by RNA-Seq. In addition, RNA-Seq identified 43% more IGH disease clones. In 69 patients lacking sensitive IGH targets, targeted NGS IGH MRD showed high correlation (R = 0.93; P = 1.3 × 10-14), better relapse prediction than conventional RQ-PCR MRD using non-IGH targets. In conclusion, RNA-Seq can identify patient-specific clonal IGH junctional sequences for MRD monitoring, adding to its usefulness for molecular diagnosis in childhood B-ALL.

Stabilization of β-catenin upon B-cell receptor signaling promotes NF-kB target genes transcription in mantle cell lymphoma.

B-cell receptor (BCR) signaling pathways and interactions with the tumor microenvironment account for mantle cell lymphoma (MCL) cells survival in lymphoid organs. In several MCL cases, the WNT/β-catenin canonical pathway is activated and β-catenin accumulates into the nucleus. As both BCR and β-catenin are important mediators of cell survival and interaction with the microenvironment, we investigated the crosstalk between BCR and WNT/β-catenin signaling and analyzed their impact on cellular homeostasis as well as their targeting by specific inhibitors. β-catenin was detected in all leukemic MCL samples and its level of expression rapidly increased upon BCR stimulation. This stabilization was hampered by the BCR-pathway inhibitor Ibrutinib, supporting β-catenin as an effector of the BCR signaling. In parallel, MCL cells as compared with normal B cells expressed elevated levels of WNT16, a NF-κB target gene. Its expression increased further upon BCR stimulation to participate to the stabilization of β-catenin. Upon BCR stimulation, β-catenin translocated into the nucleus but did not induce a Wnt-like transcriptional response, i.e., TCF/LEF dependent. β-catenin rather participated to the regulation of NF-κB transcriptional targets, such as IL6, IL8, and IL1. Oligo pull down and chromatin immunoprecipitation experiments demonstrated that β-catenin is part of a protein complex that binds the NF-κB DNA consensus sequence, strengthening the idea of an association between the two proteins. An inhibitor targeting β-catenin transcriptional interactions hindered both NF-κB DNA recruitment and induced primary MCL cells apoptosis. Thus, β-catenin likely represents another player through which BCR signaling impacts on MCL cell survival.

MSIsensor-pro: Fast, Accurate, and Matched-normal-sample-free Detection of Microsatellite Instability

Microsatellite instability (MSI) is a key biomarker for cancer therapy and prognosis. Traditional experimental assays are laborious and time-consuming, and next-generation sequencing-based computational methods do not work on leukemia samples, paraffin-embedded samples, or patient-derived xenografts/organoids, due to the requirement of matched normal samples. Herein, we developed MSIsensor-pro, an open-source single sample MSI scoring method for research and clinical applications. MSIsensor-pro introduces a multinomial distribution model to quantify polymerase slippages for each tumor sample and a discriminative site selection method to enable MSI detection without matched normal samples. We demonstrate that MSIsensor-pro is an ultrafast, accurate, and robust MSI calling method. Using samples with various sequencing depths and tumor purities, MSIsensor-pro significantly outperformed the current leading methods in both accuracy and computational cost. MSIsensor-pro is available at https://github.com/xjtu-omics/msisensor-pro and free for non-commercial use, while a commercial license is provided upon request.

Concordance of copy number abnormality detection using SNP arrays and Multiplex Ligation-dependent Probe Amplification (MLPA) in acute lymphoblastic leukaemia

In acute lymphoblastic leukaemia, MLPA has been used in research studies to identify clinically relevant copy number abnormality (CNA) profiles. However, in diagnostic settings other techniques are often employed. We assess whether equivalent CNA profiles are called using SNP arrays, ensuring platform independence. We demonstrate concordance between SNP6.0 and MLPA CNA calling on 143 leukaemia samples from two UK trials; comparing 1,287 calls within eight genes and a region. The techniques are 99% concordant using manually augmented calling, and 98% concordant using an automated pipeline. We classify these discordant calls and examine reasons for discordance. In nine cases the circular binary segmentation (CBS) algorithm failed to detect focal abnormalities or those flanking gaps in IKZF1 probe coverage. Eight cases were discordant due to probe design differences, with focal abnormalities detectable using one technique not observable by the other. Risk classification using manually augmented array calling resulted in four out of 143 patients being assigned to a different CNA risk group and eight patients using the automated pipeline. We conclude that MLPA defined CNA profiles can be accurately mirrored by SNP6.0 or similar array platforms. Automated calling using the CBS algorithm proved successful, except for IKZF1 which should be manually inspected.

TP53 and PTEN mutations were shared in concurrent germ cell tumor and acute megakaryoblastic leukemia

BackgroundThe occurrence of a mediastinal germ cell tumor (GCT) and hematological malignancy in the same patient is very rare. Due to its rarity, there have been only two reports of the concurrent cases undergoing detailed genetic analysis with whole-exome sequencing (WES), and the possible clonal relationship between the both tumors remained not fully elucidated.MethodsWe performed whole-exome sequencing analysis of mediastinal GCT and acute myeloid leukemia (AML) samples obtained from one young Japanese male adult patient with concurrent both tumors, and investigated the possible clonal relationship between them.ResultsSixteen somatic mutations were detected in the mediastinal GCT sample and 18 somatic mutations in the AML sample. Mutations in nine genes, including TP53 and PTEN both known as tumor suppressor genes, were shared in both tumors.ConclusionsAll in our case and in the previous two cases with concurrent mediastinal GCT and AML undergoing with whole-exome sequencing analysis, TP53 and PTEN mutations were commonly shared in both tumors. These data not only suggest that these tumors share a common founding clone, but also indicate that associated mediastinal GCT and AML harboring TP53 and PTEN mutations represent a unique biological entity.

Single-cell analysis of structural variations and complex rearrangements with tri-channel processing.

Structural variation (SV), involving deletions, duplications, inversions and translocations of DNA segments, is a major source of genetic variability in somatic cells and can dysregulate cancer-related pathways. However, discovering somatic SVs in single cells has been challenging, with copy-number-neutral and complex variants typically escaping detection. Here we describe single-cell tri-channel processing (scTRIP), a computational framework that integrates read depth, template strand and haplotype phase to comprehensively discover SVs in individual cells. We surveyed SV landscapes of 565 single cells, including transformed epithelial cells and patient-derived leukemic samples, to discover abundant SV classes, including inversions, translocations and complex DNA rearrangements. Analysis of the leukemic samples revealed four times more somatic SVs than cytogenetic karyotyping, submicroscopic copy-number alterations, oncogenic copy-neutral rearrangements and a subclonal chromothripsis event. Advancing current methods, single-cell tri-channel processing can directly measure SV mutational processes in individual cells, such as breakage-fusion-bridge cycles, facilitating studies of clonal evolution, genetic mosaicism and SV formation mechanisms, which could improve disease classification for precision medicine.

Inside the USCAP Journals

Inside the USCAP Journals

Comprehensive profiling of disease-relevant copy number aberrations for advanced clinical diagnostics of pediatric acute lymphoblastic leukemia.

Acute lymphoblastic leukemia is the most common pediatric cancer characterized by a heterogeneous genomic landscape with copy number aberrations occurring at various stages of pathogenesis, disease progression, and treatment resistance. In this study, disease-relevant copy number aberrations were profiled in bone marrow samples of 91 children with B- or T-cell precursor acute lymphoblastic leukemia using digital multiplex ligation-dependent probe amplification (digitalMLPATM). Whole chromosome gains and losses, subchromosomal copy number aberrations, as well as unbalanced alterations conferring intrachromosomal gene fusions were simultaneously identified with results available within 36 hours. Aberrations were observed in 96% of diagnostic patient samples, and increased numbers of copy number aberrations were detected at the time of relapse as compared with diagnosis. Comparative scrutiny of 24 matching diagnostic and relapse samples from 11 patients revealed three different patterns of clonal relationships with (i) one patient displaying identical copy number aberration profiles at diagnosis and relapse, (ii) six patients showing clonal evolution with all lesions detected at diagnosis being present at relapse, and (iii) four patients displaying conserved as well as lost or gained copy number aberrations at the time of relapse, suggestive of the presence of a common ancestral cell compartment giving rise to clinically manifest leukemia at different time points during the disease course. A newly introduced risk classifier combining cytogenetic data with digitalMLPATM-based copy number aberration profiles allowed for the determination of four genetic subgroups of B-cell precursor acute lymphoblastic leukemia with distinct event-free survival rates. DigitalMLPATM provides fast, robust, and highly optimized copy number aberration profiling for the genomic characterization of acute lymphoblastic leukemia samples, facilitates the decipherment of the clonal origin of relapse and provides highly relevant information for clinical prognosis assessment.

Cohesin-dependent regulation of gene expression during differentiation is lost in cohesin-mutated myeloid malignancies

AbstractCohesin complex disruption alters gene expression, and cohesin mutations are common in myeloid neoplasia, suggesting a critical role in hematopoiesis. Here, we explore cohesin dynamics and regulation of hematopoietic stem cell homeostasis and differentiation. Cohesin binding increases at active regulatory elements only during erythroid differentiation. Prior binding of the repressive Ets transcription factor Etv6 predicts cohesin binding at these elements and Etv6 interacts with cohesin at chromatin. Depletion of cohesin severely impairs erythroid differentiation, particularly at Etv6-prebound loci, but augments self-renewal programs. Together with corroborative findings in acute myeloid leukemia and myelodysplastic syndrome patient samples, these data suggest cohesin-mediated alleviation of Etv6 repression is required for dynamic expression at critical erythroid genes during differentiation and how this may be perturbed in myeloid malignancies.

Determination and analysis of radio-nuclei in leukaemia blood samples using SAM940TM detector

Radiation pollution becomes a major problem threatening life on the earth that causes many of the serious diseases, through acute or chronic effects. The leukemia this considered to be the most serious and common disease due to the increase of radiation pollution levels. In this study, the province of Basrah was selected because this province contains many of the remnants of the wars due to the Gulf Wars (1991, 2003). The aim of this study is to investigate the possibility of any a relationship between the radioactivity level and leukemia incidence; there are a direct or indirect relationship between the cancer rate and the exposure to radiation. Therefore, the levels of different radionuclides in leukaemic patients. In order to achieve our goal 75 samples were study and many techniques are utilized, such as the SAM 940 TM (Gamma-ray Spectroscopy). In addition, to compare between the healthy and Leukaemic samples, the UV-Vis spectrometer has been used. The results showed that there is a big difference between the radionuclide’s concentrations of the healthy and patients samples. For example, the 238U concentration in several Leukaemic patients were (2.0958 Bq/Kg), whilst at healthy individuals were (0.408 Bq/Kg).

Use of polyvinyl alcohol for chimeric antigen receptor T-cell expansion

Serum albumin has long been an essential supplement for ex vivo hematopoietic and immune cell cultures. However, serum albumin medium supplements represent a major source of biological contamination in cell cultures and often cause loss of cellular function. As serum albumin exhibits significant batch-to-batch variability, it has also been blamed for causing major issues in experimental reproducibility. We recently discovered the synthetic polymer polyvinyl alcohol (PVA) as an inexpensive, Good Manufacturing Practice-compatible, and biologically inert serum albumin replacement for ex vivo hematopoietic stem cell cultures. Importantly, PVA is free of the biological contaminants that have plagued serum albumin-based media. Here, we describe that PVA can replace serum albumin in a range of blood and immune cell cultures including cell lines, primary leukemia samples, and human T lymphocytes. PVA can even replace human serum in the generation and expansion of functional chimeric antigen receptor (CAR) T cells, offering a potentially safer and more cost-efficient approach for this clinical cell therapy. In summary, PVA represents a chemically defined, biologically inert, and inexpensive alternative to serum albumin for a range of cell cultures in hematology and immunology.

Directional association test reveals high-quality putative cancer driver biomarkers including noncoding RNAs

BackgroundMost statistical methods used to identify cancer driver genes are either biased due to choice of assumed parametric models or insensitive to directional relationships important for causal inference. To overcome modeling biases and directional insensitivity, a recent statistical functional chi-squared test (FunChisq) detects directional association via model-free functional dependency. FunChisq examines patterns pointing from independent to dependent variables arising from linear, non-linear, or many-to-one functional relationships. Meanwhile, the Functional Annotation of Mammalian Genome 5 (FANTOM5) project surveyed gene expression at over 200,000 transcription start sites (TSSs) in nearly all human tissue types, primary cell types, and cancer cell lines. The data cover TSSs originated from both coding and noncoding genes. For the vast uncharacterized human TSSs that may exhibit complex patterns in cancer versus normal tissues, the model-free property of FunChisq provides us an unprecedented opportunity to assess the evidence for a gene’s directional effect on human cancer.ResultsWe first evaluated FunChisq and six other methods using 719 curated cancer genes on the FANTOM5 data. FunChisq performed best in detecting known cancer driver genes from non-cancer genes. We also show the capacity of FunChisq to reveal non-monotonic patterns of functional association, to which typical differential analysis methods such as t-test are insensitive. Further applying FunChisq to screen unannotated TSSs in FANTOM5, we predicted 1108 putative cancer driver noncoding RNAs, stronger than 90% of curated cancer driver genes. Next, we compared leukemia samples against other samples in FANTOM5 and FunChisq predicted 332/79 potential biomarkers for lymphoid/myeloid leukemia, stronger than the TSSs of all 87/100 known driver genes in lymphoid/myeloid leukemia.ConclusionsThis study demonstrated the advantage of FunChisq in revealing directional association, especially in detecting non-monotonic patterns. Here, we also provide the most comprehensive catalog of high-quality biomarkers that may play a causative role in human cancers, including putative cancer driver noncoding RNAs and lymphoid/myeloid leukemia specific biomarkers.

PTEN and AKT1 Variations in Childhood T-Cell Acute Lymphoblastic Leukemia

Objective:PTEN/AKT pathway deregulations have been reported to be associated with treatment response in acute leukemia. This study examined pediatric T-cell acute lymphoblastic leukemia (T-ALL) samples for PTEN and AKT1 gene variations and evaluated the clinical findings.Materials and Methods:Fifty diagnostic bone marrow samples of childhood T-ALL cases were investigated for the hotspot regions of the PTEN and AKT1 genes by targeted next-generation sequencing.Results:A total of five PTEN variations were found in three of the 50 T-ALL cases (6%). Three of the PTEN variations were first reported in this study. Furthermore, one patient clearly had two different mutant clones for PTEN. Two intronic single-nucleotide variations were found in AKT1 and none of the patients carried pathogenic AKT1 variations.Conclusion:Targeted deep sequencing allowed us to detect both low-level variations and clonal diversity. Low-level PTEN/AKT1 variation frequency makes it harder to investigate the clinical associations of the variants. On the other hand, characterization of the PTEN/AKT signaling members is important for improving case-specific therapeutic strategies.

Reversible DNA Hypermethylation of the Interleukin-15 (IL-15) Promoter Induces IL-15 Expression, Drives the Pathogenesis of T-Cell Large Granular Lymphocytic Leukemia and Provides a Potential Therapeutic Approach Using 5-Azacitidine

Background and Rationale: T-cell large granular lymphocytic leukemia (T-LGLL) is an incurable clonal proliferation of CD8+ memory T-cells that leads to profound neutropenia and anemia with limited treatment options. The primary driver of T-LGLL is overexpression of interleukin-15 (IL-15), a gamma-chain cytokine. Previously, we have demonstrated that mice overexpressing IL-15 develop DNA hypermethylation and chromosomal instability that leads to the spontaneous development of LGLL (Mishra et al. Cancer Cell 2012). Further, the IL-15 promoter is known to be hypermethylated in cutaneous T-cell lymphoma (CTCL), another IL-15 driven malignancy (Mishra et al. Cancer Discovery 2016). In CTCL patients, the counterintuitive increase in IL-15 mRNA was due to hypermethylation of its promoter at the repressor binding sequences in the IL-15 gene. However, the methylation status of the IL-15 promoter in T-LGLL patients remains unknown. Concept: We hypothesize that the IL-15 promoter is hypermethylated in patients with T-LGLL, leading to aberrant overexpression of IL-15 and that this hypermethylation is a critical event in the leukemogenesis of T-LGLL. If true, demethylation of the IL-15 promoter with a resultant decrease in IL-15 transcripts should lead to apoptosis of T-LGLL cells. Hypomethylation of the IL-15 promoter, therefore, may provide a novel therapeutic approach to inhibiting IL-15, the primary driver of T-LGLL. Results: CD3+/CD8+/CD5-/dim T-cells were purified from peripheral blood of LGLL patient (n=3) and normal donor (ND) (n=3) by flow cytometry sorting. We analyzed DNA methylation and gene expression profiling using reduced representation bisulfite and RNA sequencing. With bioinformatics analysis, we determined differential methylation (1-way ANOVA P= 0.0178) and expression (1-way ANOVA P =0.0059). These data sets revealed significant differential hypermethylation of gene promoters in leukemic samples, compared to controls (Figure 1A). Reduced representation bisulfite sequencing that can identify differentially methylated regions at single base-pair resolutions demonstrated an increase in DNA methylation of the IL-15 promoter in patient samples over controls. To determine the functional significance of this finding, we treated the MOTN-1 T-LGLL cell line in vitro with the hypomethylating agent, 5-azacytidine (5-aza) at concentrations of 0.5 uM, 1 uM, 2.5 uM, and 5 uM. At 24 and 48 hours, a marked decrease in the viability of T-LGLL cells was observed, from 100% to 49.50%, p=0.037; particularly at higher concentrations of 5-aza (100% to 27% +11.30%, p=0.0030). Next, we sought to determine whether 5-aza induced hypomethylation of the IL-15 promoter. IL-15 gene expression in MOTN-1 T-LGLL cells treated with 5-aza was measured in comparison to control treated MOTN-1 cells. A marked decrease in IL-15 expression was observed at all concentrations of 5-aza compared to control (Figure 1B, p=0.0001). These results confirm that 5-aza leads to decreased transcription of the IL-15 gene, possibly due to hypomethylation of the IL-15 promoter. Finally, to determine whether a decrease in IL-15 alone was the cause of increased apoptosis of T-LGLL cells, we exposed MOTN-1 cells to a novel IL-15 inhibitor, IBI-15, and compared cell viability against MOTN-1 cells exposed to an inactive control, IBI-40. Even more profound decrease in cell viability was observed utilizing IBI-15 that targets the binding of IL-15 to its receptor (Figure 1C). Together, these data suggest that hypermethylation of the IL-15 promoter is critical to the pathogenesis of T-LGLL, and that treatment with 5-aza is sufficient to induce hypomethylation of the IL-15 promoter, decrease IL-15 transcription, and induce apoptosis in T-LGLL cells. Conclusions: Hypermethylation of the IL-15 promoter, with subsequent increase in IL-15, is critical to the pathogenesis of T-LGLL. Inhibition of the IL-15 promoter hypermethylation by 5-aza leads to down-regulation of the IL-15 gene transcript, which is sufficient to induce apoptosis of T-LGLL cells. These data suggest that 5-aza induced hypomethylation may be a novel method to induce IL-15 inhibition and a potentially efficacious clinical strategy against T-LGLL. Disclosures Brammer: Bioniz Therapeutics, Inc.: Research Funding; Viracta Therapeutics, Inc.: Research Funding; Verastem, Inc: Research Funding. Porcu:Daiichi: Research Funding; BeiGene: Other: Scientific Board, Research Funding; Spectrum: Consultancy; Viracta: Honoraria, Other: Scientific Board, Research Funding; Innate Pharma: Honoraria, Other: Scientific Board, Research Funding; Kyowa: Honoraria, Other: Scientific Board, Research Funding; ADCT: Research Funding; Incyte: Research Funding. OffLabel Disclosure: IBI-15 IBI-40 IL-15 inhibitor

CK2 Inhibitor CX4945 Shows Strong In Vivo Synergistic Anti-Leukemia Activity with Rapamycin in B-ALL Due to Enhanced Ikaros Mediated Repression of mTOR Pathway

Casein Kinase II (CK2) is a pro-oncogenic protein that is overexpressed in various cancers including leukemia. Ikaros encodes a DNA-binding zinc finger protein that functions as a tumor suppressor. Loss of Ikaros activity (genetic alteration and functional inactivation) in high-risk B-ALL is associated with resistance to conventional chemotherapy and a poor prognosis. Ikaros binds to the promoters and/or upstream regulatory sequences of its target genes and regulates their transcription via chromatin remodeling. CK2 phosphorylates Ikaros and impairs its tumor suppressor functions and interferes with Ikaros' ability to repress cell cycle progression and the PI3K pathway. Targeted inhibition of CK2 using specific inhibitor CX4945 restores Ikaros tumor suppressor function in high-risk B-ALL even in cases with single allele Ikaros deletion. CX4945 showed strong in vivo anti-leukemia effect in high risk B-ALL patient derived xenograft models (Song et al, Blood 2015). CX4945 is currently in clinical trial for solid tumors. mTOR is the key component of the PI3K/AKT/mTOR pathway - a major signaling pathway that stimulates cellular proliferation in B-ALL. Rapamycin is a specific inhibitor of mTOR that is currently in clinical trial for the treatment of relapsed/refractory leukemia. Here we report in vivo efficacy of combination therapy of rapamycin with CX4945 using high risk B-ALL pre-clinical models and describe mechanism of synergistic action of CX4945 and rapamycin. Methods and results: The therapeutic effect of the CX-4945, in combination with rapamycin was tested using three different patient-derived xenografts (PDX) from patients with high-risk B-ALL and its efficacy will be compared to single-drug treatment. All these leukemia samples were obtained from high risk pediatric B-ALL patients with high CK2 expression and one had IKZF1 haplo - deficiency. After engraftment following tail vein inoculation of leukemia cells into immunocompromised (NRG) mice, mice were treated with vehicle, rapamycin or CX4945 alone or combination of both CX4945 and rapamycin. Anti-tumor effect was measured by quantifying leukemia burden in bone marrow, spleen and peripheral blood by cell count and immune-phenotyping using flow cytometry. Overall survival was measured using Kaplan -Myers analysis in another cohort after similar treatment regimen. The analysis of Ikaros' global genome-wide occupancy in the human B-ALL cell line, Nalm6, and in primary cells from patient with B-ALL, showed that Ikaros binds to the upstream regulatory element (URE) of MTOR gene. Further, Ikaros binding to MTOR promoter was confirmed by quantitative chromatin immunoprecipitation (qChIP) in primary B-ALL cells. The role of Ikaros in regulating mTOR transcription in Nalm-6 B-ALL cell line was tested using gain-of-function and loss-of-function experiments. Ikaros knock-down with shRNA results in increased transcription of mTOR whereas overexpression of Ikaros by transduction of Nalm-6 cells with Ikaros-containing retrovirus resulted in sharp reduction of mTOR expression. Since Ikaros function in B-ALL is negatively regulated by pro-oncogenic Casein Kinase II (CK2), we tested whether CK2 can regulate expression of mTOR. Over-expression of CK2 via retroviral transduction resulted in increased transcription of the mTOR mRNA, as measured by qRT-PCR, as well as increased overall expression of mTOR protein, as measured by western blot. Increased expression of CK2 was associated with a loss of Ikaros binding to the MTOR gene promoter. CK2 inhibition was also associated with reduced histone H3K9ac and H3K4me3 marks at the mTOR promoter suggesting that CK2 and Ikaros regulate mTOR transcription via chromatin remodeling. In summary, these results establish in vivo anti-leukemia effect of combination therapy using rapamycin and CX4945 in high risk B-ALL PDX models. CK2 inhibition with CX4945 represses the mTOR pathway by enhancing Ikaros-mediated transcriptional repression of mTOR and PI3K pathway genes. Results provide the rationale for evaluating clinical use of CK2 inhibitors in combination with mTOR inhibitors in high risk leukemia with dysregulation of mTOR/PI3K pathway. The results of our study establish a new paradigm for targeting the transcriptional network that regulates expression of the gene in hyperactive oncogenic signaling pathways such as mTOR. Disclosures Payne: Elf Zone, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

A Novel Proteomic Profiling of the Bone Marrow Microenvironment Reveals Elevated Levels of the Chemokine CCL23 Isoforms in Acute Myeloid Leukemia

The bone marrow (BM) microenvironment is increasingly recognized as an important contributor to acute myeloid leukemia (AML) pathogenesis. However, despite growing interest in characterizing different components and cellular architecture of the BM niche and their biological significance in leukemogenesis, the proteomic constitution of the BM extracellular compartment that distinguishes a leukemic niche from its normal counterpart has not yet been fully described. We therefore performed a quantitative, large-scale proteomic analysis of 1,305 human proteins of the non-cellular compartment of BM (plasma) samples from ten relapsed or refractory AML patients and from ten age- and sex-matched healthy donors (HDs) using an aptamer-based, highly multiplexed, affinity proteomics platform (SOMAscan). This screen identified a total of 168 differentially abundant proteins, of which 91 were significantly more and 77 proteins significantly less abundant in leukemic BM compared with healthy marrow (FC ≥ 1.5, FDR ≤ 0.05). Comparative analysis of BM plasma and peripheral blood (PB) serum samples from the same AML patients and HDs revealed 65 similarly regulated proteins (37 up-regulated vs. 28 down-regulated) and 1 differently regulated protein between the two compartments. Out of the total 168 proteins, 102 proteins were specifically dysregulated only in the BM compartment. TruSeq Stranded Total RNA-sequencing (Illumina) was also performed using paired-end 75bp sequencing on a HiSeq 3000. RNA was isolated from PAXgene BM RNA tubes (Qiagen) collected in parallel with samples for proteomic analysis. Results of analysis of differentially expressed transcripts only partially overlapped with those candidates identified from our validated proteomic approach, indicating that sequencing of RNA derived from cellular sources of BM may be a suboptimal screening strategy to determine the true proteomic composition of the extracellular compartment of the AML marrow microenvironment. In addition to several previously reported proteins, our proteomics screen discovered numerous aberrantly expressed proteins in leukemic marrow whose role in AML pathogenesis is currently unknown. Using pathway analysis, we identified sets of proteins enriched for specific biological pathways including RAS, ephrin, PDGF, PI3K/AKT, MAPK, Notch, TLR, JAK-STAT, NFκB, Rap1, and Tie2 signaling pathways. A systems biology analysis approach revealed the highly connected network of cytokines and chemokines as the most striking AML-associated proteomic alteration in the BM. We identified IL-8 as a differentially expressed and key central molecule of this network in AML, consistent with recent reports. Importantly, we also identified significantly elevated levels of CKβ8 and CKβ8-1, alternatively spliced isoforms of the myelosuppressive chemokine CCL23 also known as myeloid progenitor inhibitory factor 1 (MPIF-1) or CKβ8, in both leukemic marrow and PB serum samples (Figure 1). Given the critical importance of cytopenias, often disproportional to the degree of leukemic marrow involvement, in the morbidity and mortality of patients with myelodysplastic syndrome (MDS) and AML, we subsequently confirmed this striking finding by performing orthogonal validation in a larger cohort of MDS and AML patients using an ELISA-based immunoassay. This novel finding suggests the possibility that CCL23 may play a role in suppression of normal hematopoiesis in MDS and AML. In support of this hypothesis, we demonstrated in vitro myelosuppressive effects of CCL23 isoforms on colony formation by human CD34+ hematopoietic stem and progenitor cells (HSPCs) in an in vitro colony forming unit assay, resulting in an approximately 2.5-fold decrease in CFU-GM and an evident decrease in CFU-GEMM counts. In summary, our broad and quantitative proteomic dataset of extracellular factors present in leukemic and normal aging bone marrow has already provided novel mechanistic insights into AML pathogenesis and should serve, together with paired RNA-sequencing information, as a useful public resource for the research community. Disclosures Lai: Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Speakers Bureau; Astellas: Speakers Bureau; Daiichi-Sankyo: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees. Hourigan:SELLAS Life Sciences Group AG: Research Funding; Merck, Sharpe &amp; Dohme: Research Funding.

Molecular Classification of Small B-Cell Lymphomas Using Digital Multiplexed Gene Expression Applicable to Formalin-Fixed Paraffin-Embedded Tissues

Background: Small B-cell lymphomas encompass a group of lymphoid neoplasms with high heterogeneity. However, the lack of relatively specific diagnostic markers for most of these diseases make their diagnosis challenging. Methods: Using NanoString platform, a random forest-based molecular classification model was trained on 203 malignant and 98 non-malignant formalin-fixed paraffin-embedded (FFPE) tissues. Candidate genes were selected from microarray gene expression data of 891 small B-cell lymphomas. All malignant biopsies were derived from individuals diagnosed as a well-defined small B-cell lymphoma entity, including 17 chronic lymphocytic leukemia/small lymphocytic B-cell lymphoma (CLL/SLL), 74 follicular lymphoma (FL), 34 classical mantle cell lymphoma (cMCL), 5 non-nodal mantle cell lymphoma (nnMCL), 56 mucosa-associated lymphoid tissue lymphoma (MALT), 3 splenic marginal zone lymphoma (SMZL), and 14 nodal marginal zone lymphoma (NMZL). A leave-one-out prediction (CLL/SLL, FL, MCL, MZL, and non-malignancy) was applied to test the accuracy of prediction of the model, and Gini Index of each gene was taken into consideration in simplifying the molecular assay applicable to routine test. Results: A total of 154 candidate genes were initially selected for training the molecular classification model. Genes were initially selected for training the molecular classification model. Thirty-two genes were specifically highly expressed in a single subgroup with Gini Index more than 0.001. The most important genes for classification were CCND1, ZBTB32, MME, BHLHE41, SOX11, and RGS13. The most distinguishable entity was CLL/SLL (ZBTB32, CLNK, and CD200), followed by cMCL/nnMCL (CCND1 and SOX11), and FL (MME, RGS13, and ELL3). Using the molecular classification model trained on the most specific 33 genes, the consistency of diagnosis were 97.5%. Conclusions: In conclusion, our molecular model robustly recognized different subtypes of small B-cell lymphomas in leukemic FFPE samples, which provides a novel approach to precise diagnosis and classification of small B-cell lymphomas. Figure Disclosures Zhou: Nanjing Iaso Biotherapeutics Co. Ltd.: Other: Chairman of Advisory Committee of Science and Medicine.