ALBERT

Description: Chronic lymphocytic leukemia (CLL), characterized by the progressive and uncontrolled accumulation of CD19+ B cells, currently remains as an incurable malignancy. The difficulties of eliciting curative measures in CLL are partly driven by the adaptability of the transcriptional response mediated by epigenetic mechanisms. In this study, we sought to better characterize the complexities of the CLL transcriptional profile by conducting an integrative analysis between the B cell enhancer and super enhancer signatures defined from 3 B cell H3K27Ac ChIPseq samples (CD19+ B cell, GM12878, and MEC1), the DNA methylation signatures defined from reduced-representation bisulfite sequencing (RRBS) of 42 CLL patient and 8 healthy donor samples, and the mRNA expression signatures defined from RNA sequencing of 47 CLL patient and 5 healthy donor samples. From our analysis, we identified super enhancers (SEs) in each of the ChIPseq profiles (approximately 4% of called enhancers) and discovered 741 SEs in GM12878, 374 SEs in MEC1, and 523 SEs in the CD19+ B cell profiles, respectively. Based on MSigDB gene ontology analysis, many of the genes corresponding with SEs were involved in pathways regulating immune signaling activation (e.g. TNFA_SIGNALING_VIA_NFKB, INFLAMMATORY RESPONSE) or metabolic homeostasis (e.g. MTORC1_SIGNALING, FATTY_ACID_METABOLISM). By further analyzing the corresponding expression level of SE-associated genes in CLL patients, we identified 190 transcripts associated with SEs that were significantly overexpressed in CLL patient B cells (Student's t-test p

Description: Recent studies have shown that overexpression of Heat Shock Factor (HSF) 1 in aneuploid tumor cells can overcome deficiencies in heat shock protein (HSP) 90-mediated protein folding and restore protein homeostasis. In this study we determined the mechanisms by which HSF1 promotes HSP90 function and CLL pathogenesis using CLL as model system. We report that HSF1 is overexpressed in CLL and treatment with triptolide (a small molecule inhibitor of HSF1) induces apoptosis in cultured and primary CLL B-cells. Mechanistically, we demonstrate that knockdown of HSF1 or its inhibition with triptolide results in the reduced association of HSP90 with its kinase co-chaperone cell division cycle 37 (CDC37), leading to the partial depletion of HSP90 client kinases, Bruton's Tyrosine Kinase (BTK), c-RAF and cyclin-dependent kinase 4 (CDK4). RNA-sequencing of control and HSF1-knockdown CLL cells revealed that HSF1 regulates the transcription of upstream modulators of the NF-kB pathway including MYD88 and TLR1. Consequently, treatment with triptolide or knockdown of HSF1 inhibits NF-kB signaling in CLL cells. In an in vivo model of CLL, tail vein injection of luciferase-expressing Mec-1 cells into Rag2-/-IL2Rγc-/- mice followed by daily intraperitoneal injection of minnelide (a pro-drug of triptolide) for 28 days reduced in vivo disease burden and conferred significant survival advantage (p

Description: The polycomb repressive complex (PRC) 2 contains 3 core proteins, EZH2, SUZ12, and EED, in which the SET (suppressor of variegation–enhancer of zeste-trithorax) domain of EZH2 mediates the histone methyltransferase activity. This induces trimethylation of lysine 27 on histone H3, regulates the expression of HOX genes, and promotes proliferation and aggressiveness of neoplastic cells. In this study, we demonstrate that treatment with the S-adenosylhomocysteine hydrolase inhibitor 3-deazaneplanocin A (DZNep) depletes EZH2 levels, and inhibits trimethylation of lysine 27 on histone H3 in the cultured human acute myeloid leukemia (AML) HL-60 and OCI-AML3 cells and in primary AML cells. DZNep treatment induced p16, p21, p27, and FBXO32 while depleting cyclin E and HOXA9 levels. Similar findings were observed after treatment with small interfering RNA to EZH2. In addition, DZNep treatment induced apoptosis in cultured and primary AML cells. Furthermore, compared with treatment with each agent alone, cotreatment with DZNep and the pan-histone deacetylase inhibitor panobinostat caused more depletion of EZH2, induced more apoptosis of AML, but not normal CD34+ bone marrow progenitor cells, and significantly improved survival of nonobese diabetic/severe combined immunodeficiency mice with HL-60 leukemia. These findings indicate that the combination of DZNep and panobinostat is effective and relatively selective epigenetic therapy against AML cells.

Description: Background Successful allogeneic stem cell transplantation (HSCT) in treatment of patients (pts) with acute myeloid leukemia (AML) is dependent upon graft-versus-leukemia, suggesting that intact immune surveillance is essential for eradicating minimal residual disease. Myeloblast-induced T-cell tolerance through overexpression of indoleamine 2,3-dioxygenase (IDO) is thought to play a significant role in immune evasion through upregulation of tryptophan (Trp) catabolism and kynurenine production, resulting in a Trp-poor environment that leads to immune system suppression. Indoximod is a small-molecule inhibitor of the IDO pathway that acts directly on immune cells to reverse IDO pathway-mediated suppression. We are assessing the safety and preliminary efficacy of indoximod in combination with standard induction chemotherapy in patients (pts) with newly diagnosed AML. Methods In this open-label, multicenter, phase 1 study (NCT02835729), eligible pts with newly diagnosed AML were treated with indoximod in combination with induction chemotherapy (idarubicin 12 mg/m2/d x3 days with cytarabine 100 mg/m2/d x7 days). Using a "3+3" design, indoximod (600 mg [dose level 0], 1000 mg [dose level 1], 1200 mg [dose level 2]) was given orally every 8 hours (Q8h) starting on day 9 of induction. Regimen limiting toxicity (RLT) was defined as any ≥ grade 3 non-hematologic adverse event (AE) that was not incontrovertibly related to the underlying AML or cytarabine or idarubicin. After induction, pts received up to 4 cycles of high dose cytarabine (HiDAC) consolidation while continuing indoximod. Patients continued on maintenance indoximod for up to 6 months from completion of consolidation therapy. Indoximod was discontinued 4 weeks prior to HSCT in eligible patients and not restarted as maintenance post-HSCT. Results As of July 15, 2018, 31 pts were enrolled (median age 55 years, range 18-78; 77% male). Six patients did not proceed with study therapy due to a diagnosis of acute promyelocytic leukemia, issues with medical insurance coverage, consent withdrawal, critical illness, and intestinal myeloid sarcoma preventing oral intake. Pts who received ≥1 dose of indoximod were included in the intention-to-treat (ITT) analysis (n=25), and pts who received ≥80% of their scheduled indoximod doses were included in the per-protocol (PP) analysis (n=19). Reasons for not completing ≥80% of indoximod doses were: consent withdrawal (n=3), inability to swallow (n=2) and physician decision (n=1). Of the 19 PP patients, 16 (84%) had either unfavorable karyotype or adverse mutation profile and 3 (16%) had secondary AML (s-AML). Indoximod combined with induction chemotherapy was well tolerated; no RLT was observed. The most frequent grade ≥3 non-hematologic treatment-emergent AEs in the ITT population, regardless of attribution, were febrile neutropenia (60%), hypoxia (16%), atrial fibrillation (12%), pneumonia (12%), hypocalcemia (12%), and hypotension (12%). Among 25 ITT pts, 21 (84%) achieved a remission (CR/CRh/CRi/CRp), and 15 of 19 (79%) in the PP analysis achieved remission. Among 12 pts with measurable residual disease (MRD) available in remission, 10 (83%) had MRD

Description: Background: Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults in the Western countries but is relatively rare in East Asia. Mutations in myeloid differentiation primary response gene 88 (MYD88) occur in 2.0-4.4% of Caucasian patients with CLL. However, Asian subjects showed a higher MYD88 mutation frequency of 8% and MYD88 mutations were associated with unfavorable prognosis in Asian CLL patients with mutated IGHV gene. To further explore the molecular and pathological mechanisms of the MYD88 L265P mutation in CLL, we performed a pilot study to investigate the transcriptome-wide differential expression between CLL patients with and without MYD88 L265P mutation. Methods: mRNA sequencing was performed on a cohort of 15 Chinese CLL patients with MYD88 L265P mutation (n=5) or wild-type MYD88 gene (n=10). Differential expression and pathway enrichment analyses were performed based on FPKM values after processing the raw sequencing reads. The alternative splicing events were analyzed using JuncBASE and the differences in splicing patterns were compared between the two groups. The MYD88 L265P and wild-type genes were overexpressed in the MEC-1 cell line using lentiviral vectors, and stable cell lines were established after puromycin selection. Real time quantitative PCR was used to determine the mRNA expression of spliceosome pathway genes. The protein expression of TLR pathway was assessed with Western Blot. The PrestoBlue™ assay was used for determining IC-50 of spliceosome inhibitor-treated CLL cell lines, and flow cytometry was utilized for apoptosis and cell death analyses. Results: mRNA sequencing analysis revealed that 4231 genes showed significant differences in mRNA expression between MYD88 mutant and wild-type CLL patients. Of these genes, 3033 were up regulated and 1198 were down regulated in patients with MYD88 L265P mutation. Pathway analysis demonstrated that tumor necrosis factor (TNF) signaling, cell cycle, spliceosome, ribosome, p53 signaling, and NF-κB signaling pathways were significantly enriched among differentially expressed genes. Interestingly, about two thirds (19 out of 30) of differentially expressed genes in the spliceosome pathway were down regulated in CLL samples with MYD88 mutation. Quantitative RT-PCR confirmed the down regulation of spliceosome genes including SF3A1, SF3A2, SF3B4, SF3B5 and PUF60 in MYD88 mutated samples in an independent CLL patient cohort. Analysis of alternative splicing events revealed a total of 180 genes with inter-group splicing differences, among which differential splicing events were most common in the genes involved in immune system regulation, i. e. CD22, CD79B, CD200 and IKZF1 etc. The MEC1 cells stably overexpressing MYD88 L265P demonstrated decreases in RelA and TRAF6 expression as compared to wild type MYD88-overexpressing cells, which was in agreement with RNA-seq analysis results in CLL patient samples. In addition, we found that overexpression of MYD88 L265P mutant resulted in decreased mRNA expression of several spliceosome genes such as SF3A1, SF3A2, SF3B4, SF3B5 and PUF60 in the stable cell line. Furthermore, MYD88 L265P increased the sensitivity of MEC1 cells to spliceosome inhibitor sudemycin D6 (SD6) by 3 fold, while reducing their sensitivity to Ibrutinib by two fold. Conclusion: We discovered the differential mRNA expression landscapes related to MYD88 mutation status in CLL and their contributions to the progression of CLL through multiple pathways. Of particular interest was the down regulation of spliceosome pathway genes. In a CLL cell line model, we found that, compared with the MYD88 wild-type CLL cell line, CLL cells overexpressing the mutant MYD88 were more sensitive to spliceosome inhibitor SD6, suggesting that inhibiting spliceosome function may be a potential therapeutic target for CLL patients with mutant MYD88 gene. In summary, our findings provide novel insights for further understanding of the molecular and pathological mechanisms of MYD88 mutation in CLL. Disclosures Lagisetti: St. Jude Children's Research Hospital: Patents & Royalties: Chandraiah Lagisetti is listed as inventors on patents assigned to St. Jude Children's Research Hospital covering sudemycin D6 and was employed by SRI Biosciences at the time these experiments were carried out. Webb:St. Jude Children's Research Hospital: Patents & Royalties: Thomas R. Webb is listed as inventors on patents assigned to St. Jude Children's Research Hospital covering sudemycin D6 and was employed by SRI Biosciences at the time these experiments were carried out..

Description: Background: Most cancer deaths are caused by hematogenous spread and subsequent metastasis. Emerging data indicates that the presence of circulating tumor cells in peripheral blood (CTC) and disseminating tumor cells in bone marrow (DTC) is an early event in tumorigenesis. These circulating or disseminating rare tumor cells may represent a distinct clone with cancer stem cell properties (metastatic stem cells). Clinically, the presence of CTC and DTC is relevant to overt metastases. Early detection and characterization of these rare biologically distinct tumor cells with sensitive and specific methods provide vital information for cancer biology and early clinical intervention and thus improve patients’ survival. Method Design: Genomic DNA was extracted from patient blood, bone marrow aspirate specimens and cancer cell lines prior to digestion with 4 methylation sensitive restriction enzymes. Hypermethylated CpG island regions in tumors, in contrast to their counterpart in normal cells which are completely digested, remain resistant to digestion and therefore can be differentially amplified by PCR. Thus, the specific PCR products on the gel represent the specific methylation loci in the tumor cells in the patient specimens. Results: First, we tested a total of 26 cancer cell lines including 17 hematopoietic tumors and 9 solid tumors. The hematopoietic tumor cell lines represent a spectrum of B-cell malignancies, T-cell lymphoblastic leukemia and acute myeloid leukemia. The solid tumor cell lines represent the major anatomic sites of human carcinoma including lung, colon, breast, prostate, ovarian and skin (melanoma). We found multiple DNA methylation markers specific for lymphoid, myeloid and solid tumors. By using DLC-1 (deleted in liver cancer-1), a tumor suppressor gene, as a single marker, CpG island methylation was detected in 13 out of 17 hematopoietic tumor cell lines (76%) and 6 out of 9 solid tumor cell lines (67%). We then used B-cell acute lymphoblastic leukemia (B-ALL) as a testing case to verify the method in a total of 135 clinical specimens. DLC-1 methylation was detected in 64% and 54% of diagnostic bone marrow aspirates and peripheral blood specimens, respectively, but none was detected in normal or non-leukemic bone marrow or blood control samples. By adding two additional methylation markers PCDHGA12 and CDH1, greater than 90% of B-ALL patients can be detected. We also traced 4 B-ALL cases and 4 follicular lymphoma cases up to 10 years retrospectively and found that the DLC-1 methylation is exactly correlated with patient clinical status. Lastly, by mixing normal and leukemic cell genomic DNA, analytic sensitivity was determined as 0.1% or 10−3 in a single-step PCR and 0.00001% or 10−6 in a nested PCR suggesting that this method is capable of detecting as few as 5 leukemic cells in a single-step PCR. Conclusion: By utilizing tumor specific DNA methylation marker(s), we have developed a simple, highly sensitive and specific gel-based PCR assay, namely Multiple Methylation Sensitive Enzyme Restriction PCR (MSR-PCR) for detection of CTC and DTC from blood and bone marrow of majority of hematopoietic malignancies. The method can potentially be used for early diagnosis and molecular monitoring in vast majority of clinical cancer patients.