ALBERT

Description: Janus kinases (JAK) comprise a small family of cytoplasmic protein tyrosine kinases, which play an important role in the initiation of cytokine-triggered signaling events via signal transducer and activator of transcription (STAT) proteins. The recent reports of an activating somatic mutation in codon 617 of the JAK2 gene (JAK2V617F mutation) in patients with myeloproliferative disorders (MPDs), has opened new avenues for the development of targeted therapies for these malignancies and clinical trials with JAK2 inhibitors are underway. We report here the activity of Atiprimod (N,N-diethyl-8-dipropyl-2-azaspiro[4,5]decane-2-propanamine), a novel compound with anti-inflammatory properties, in retrovirus-transduced JAK2V617F mutant-expressing murine FDCP-EpoR cells, set-2 cells, and blood cells from patients with polycythemia vera (PV). We compared the growth inhibitory effect of Atiprimod against two mouse FDCP cell lines transfected with erythropoietin receptor (Epo-R), and either wild-type JAK2WT or mutant JAK2V617F, and human megakaryoblastic leukemia cells with mutated JAK2V617F (set-2 cells). The growth inhibitory effect was assessed using 3-days MTS assay. Atiprimod was more potent against FDCP cells carrying mutant JAK2V617F cells (IC50 0.42 μM) and set-2 cells (IC50 0.53 μM) than FDCP wildtype JAK2WT cells (IC50 0.69 μM). Atiprimod inhibited the phosphorylation of JAK2 and downstream STAT3, STAT5, and AKT proteins in a dose- and time-dependent manner. It induced apoptosis, as evidenced by increase in mitochondrial membrane potential, caspase3 activity, and cleavage of PARP protein. The anti-proliferative effect on expanded PV patient progenitor’s cells was paralleled by a decrease in JAK2V617F mutant allele frequency in BFU-E or CFU-GM clones in clonogenic assay. However, co-culturing of JAK2V617F mutant cells with three different bone marrow stromal cell lines (Hs5, ABM-MSC, NK-Tert) either directly (cell on cell) or indirectly (separated by 0.4 μm micropore membranes) for 48 hours resulted in a significant protection of mutant cells from the effect of Atiprimod. Co-culturing of bone marrow stromal cells prevented Atiprimod (0.4 and 0.8 μM) induced apoptosis, and reversed the inhibition of phosphorylation of STAT proteins. Our results suggest that cytokines secreted by stromal cells might play an important role in protecting the hematopoietic cells from a JAK2 inhibitor. Further dissection of the nature of interactions between JAK2V617F mutant cells and marrow stromal cells may lead to new therapeutic avenues for patients with MPD.

Description: The dominant gain-of-function mutation in the tyrosine kinase JAK2 (1849 G to T) has been detected in over 80% PV patients and in approximately half of ET or PMF patients. The mutation results in a non-synonymous amino acid substitution at position 617 (valine to phenylalanine) in the JH2 pseudo-kinase auto-inhibitory domain. Current models predict that expression of mutant JAK2V617F in patient hematopoietic stem cells leads to activation of signaling pathways important for proliferation and survival. Furthermore, experimental evidence using murine models, in which JAK2V617F is retrovirally over-expressed, display a phenotype that closely resembles PV, including progression to myelofibrosis. Previous reports have indicated that “in vitro” expansion of PV progenitors favors erythroid precursors with decreased JAK2V617F mutational burden (Exp. Hematol. (2007) 35(4): 587–95). Investigators in these studies, however, did not report changes in JAK2V617F frequency in sorted live erythroid progenitors after “in vitro” expansion. Furthermore, CD34+cells from PV patients cultured in the presence of 20% serum indicated an inverse relationship between JAK2V617F frequency and Epo dose (Blood (2006) 108(9):3128–34). However, these reports failed to correlate Epo dose, and JAK2V617F frequency, with live “in vitro” expanded purified erythroid progenitor cells. Using our published procedure (Exp. Hematol. (2007) 35(4): 587–95), we expanded erythroid progenitors from healthy volunteers and JAK2V617F positive PV patients (mutational burden ranging from 20–90%) in serum-free media supplemented with SCF, Tpo, and Flt3L (100 ng/mL each - week 1) followed by SCF (50 ng/mL), IGF1 (50 ng/mL), and Epo (0, 0.003, 0.03, 0.3, and 3 Units/mL - week 2). Progression of progenitor expansion, erythroid lineage commitment, live/dead cells, and JAK2 mutation frequency was followed by flow cytometry and real-time PCR. At the end of week 2, cells were harvested, stained with anti-CD71 (transferrin receptor), anti-CD235A (glycophorin A), and the Aqua Live/Dead cell reagent. Stained cells were fixed in 2% paraformaldehyde, analyzed, and sorted on a FACSAria flow cytometer. Live, expanded progenitor cells were sorted into CD235A positive and negative populations, and subsequently used for estimation of JAK2 frequency by quantitative real-time PCR (Exp. Hematol. (2007) 35(1): 32–38). Our results indicate expansion and erythroid commitment of PV progenitor cells in the absence of Epo correlates with the patients’ intrinsic JAK2V617F burden. Furthermore, irrespective of JAK2V617F frequency in purified peripheral blood granulocytes, an inverse correlation between Epo dose and mutation frequency in “in vitro” expanded purified erythroid progenitors was not observed in all patients examined.

Description: Hemoglobin Monroe (Hb Monroe) results from a point mutation (G-〉C) in the last nucleotide of β globin exon 1, which is also the penultimate nucleotide of codon 30 of the β globin mRNA (AGG-〉ACG/Arg-〉Thr). Hb Monroe was described seventeen years ago simultaneously by two groups: in an African American female with β thalassemia intermedia, wherein the thalassemia was thought to result from highly unstable peptide (Hemoglobin.1989;13:67); and in a North African female with compound β thalassemia (Proc Natl Acad Sci U S A.1989;86:1041) wherein the Hb Monroe mutation was thought to result in abnormal pre-mRNA splicing as detected in an in vitro cell-free transcription assay. We evaluated a 31-year old previously asymptomatic woman of Asian Indian (Bengali) descent, who presented with flu like symptoms and found to have low hemoglobin level (9.5 gm/dL), microcytosis (MCV 68 fL), moderately elevated liver enzymes and serum ferritin concentration of 3000 ng/ml. A liver biopsy revealed increased liver iron and significant fibrosis. Hemoglobin analysis, which was interpreted as compound heterozygosity for HbE/β0 thalassemia, revealed HbF: 51%, HbE: 43.2%, HbA2: 5.8%. β globin gene sequencing showed Hb Monroe and E mutations. The asymptomatic brother of the proband had borderline anemia (Hb 12 gm %), microcytosis (MCV 70 fl), HbF: 8.5 %, HbA: 87.2%, HbA2: 5.0% and was heterozygous for Hb Monroe mutation by Bme 15801 restriction enzyme analysis of genomic DNA. We set out to determine the molecular basis of the thalassemia phenotype associated with Hb Monroe mutation and whether this mutation in our subjects is present on African haplotype or had arisen independently. Since we could not detect the mutant peptide either in fresh hemolysate or reticulocyte enriched preparations; we expanded the peripheral blood erythroid progenitor cells in vitro of both proband and her brother. Hemoglobin analysis by both HPLC and mass spectrophotometry did not detect Hb Monroe peptide in the expanded cells. β globin cDNA from the reticulocytes and expanded erythroid progenitors was amplified using three different primer sets and no splice variants were detectable. Sequencing of the amplified cDNA revealed only normal β globin mRNA transcript. Other β globin gene mutations cis to Hb Monroe are being ruled out; to date, we have not found any promoter region or stop codon mutations, deletions or splicing mutations from promoter - 90 region to 3′ UTR including poly-A region. Haplotype analysis revealed a different haplotype from the two African American patients, indicating an independent origin of Hb Monroe mutation in our cases. Interestingly, both of our cases have elevated HbF, as did the two originally reported Hb Monroe patients (16.5 and 85%) and a currently unreported African American patient with sickle cell - β0 thalassemia due to Hb Monroe (11.4%). Hb F was also high in a group of nine patients reported with sickle cell - β0 thalassemia due to Hb Monroe (3.1%– 8.9%; Hemoglobin.1998; 22:153). We conclude that this missense mutation, IVS1-1 (G-〉C/Arg 30 Thr) results in undetectable transcript and mutant Hb peptide leading to β0 thalassemia. The molecular mechanism of the undetectable mutant transcript is being investigated.

Description: Polycythemia vera (PV) is an acquired, clonal stem cell, myeloproliferative, hematological disorder with variable increase in erythrocytes, neutrophils and granulocytes. PV causes significant morbidity and mortality from thrombotic and hemorrhagic complications and has a propensity for leukemic transformation. Phlebotomy, interferon a and myelosuppressive chemotherapy have been the cornerstones of treatment to date. With no specific drugs to treat PV effectively, the development of new therapeutic modalities is important. A somatic mutation in the JAK2 tyrosine kinase (V617F) causing constitutive activation of the JAK/STAT pathway was recently reported in over 80% of PV patients. Based on this observation, we explored the therapeutic efficacy of tyrosine kinase inhibitors (TKIs) such as imatinib (Gaikwad A et al, Blood106: 2601, 2005) and AMN107 for PV. These TKIs showed marginal efficacy in vitro. Research using natural sources has led to important pharmacological targets for cancer therapy. Several plant molecules have either been introduced to the US market or are in late-phase clinical trials. We focused on a natural product called “Avicin D,” which is a plant derived triterpene electrophilic molecule with cyto-protective and anti-inflammatory functions (Haridas V et al, J. Clin. Invest113: 65, 2004; Haridas V et al, PNAS, 98:11557, 2001). Avicin D is reported to initiate selective pro-apoptotic activity in malignant cells (Mujoo K, et al. Cancer Res.61: 5486, 2001) by direct perturbation of mitochondria (Haridas V, et al. PNAS, 98: 5821, 2001). It has also been shown to down-regulate a group of pro-survival, anti-apoptotic proteins that act downstream of cytochrome c release, including HSP70, HSP90 and XIAP (Gaikwad A, et al. Clin. Cancer Res. 11: 1953Gaikwad A, et al. Clin. Cancer Res. 11: 2005). In addition, Avicin D also blocks glycolysis, a key metabolic process that malignant cells exploit for their proliferation and survival (Warburg phenomenon). To examine Avicin D’s therapeutic efficacy for PV, we utilized mouse reporter cells expressing the JAK2 V617F protein. Avicin D inhibited the growth of these cells with an IC50 of 2μM. Interestingly, in cells carrying wild-type JAK2 there was no significant inhibitory effect. In addition, Avicin D showed marked growth inhibition of human erythroleukemic cells (HEL) that harbor the JAK2 V617F mutation at an IC50 of 2μM. We then examined Avicin D’s effect on in vitro expanded native human erythroid progenitor cells. Avicin D showed specific growth inhibition of erythroid progenitor cells from PV patients (IC50 ~3μM) with no significant effect on the normal progenitors. We conclude that Avicin D should be a promising candidate for clinical trials of PV and other disorders that are associated with JAK2 V617F somatic mutations.

Description: The seminal discovery of the Bcr-Abl tyrosine kinase inhibitor, imatinib, has revolutionized the therapy of a clonal myeloproliferative disorder, chronic myeloid leukemia (CML). Polycythemia vera (PV) is also a clonal myeloproliferative disorder due to an acquired mutation of hematopoietic stem cells. The majority of PV patients have a somatic mutation of the Janus 2 tyrosine kinase (JAK2 V617F). This provides an excellent clinical opportunity for development of novel tyrosine kinase inhibitors. We studied the efficacy of tyrosine kinase inhibitors in PV and showed a limited effect of imatinib in vitro and in vivo (Gaikwad et al, Blood106: 2601, 2005). Here, we report on the effect of two tyrosine kinase inhibitors, AEE788 and AMN107 (Novartis Pharma) on PV. AEE788 causes growth arrest and apoptosis via inhibition of multiple receptor kinases, including EGFR, HER-2 and VEGFR, which are known to stimulate tumor cell growth and angiogenesis. AMN107 targets Bcr-Abl, KIT, and PDGFR and has potent activity (30 times) even against mutated Bcr-Abl variants that are resistant to imatinib. We used mouse FDCP reporter cells expressing either the wild-type JAK2 (JAK2 cells) or the mutant JAK2 V617F protein (V617F cells) to examine the growth inhibitory activity of these drugs. AEE788 specifically inhibited the growth of V617F cells at an IC50 of 1μM with no effect on wild type JAK2 cells. We also tested the effect of AEE788 on human erythroleukemic cells (HEL) that carry the JAK2 V617F mutation. AEE788 markedly inhibited the growth of HEL cells with an IC50 ~0.8μM. In contrast, AMN107 showed only marginal growth inhibition on V617F cells (IC50 of 12μM) and on HEL cells (IC50 of 8μM). The effect of AMN107 was similar to imatinib activity, wherein only high concentrations of imatinib (IC50 of 5μM) would inhibit the V617F cells, in contrast to imatinib’s known growth inhibitory effect at lower concentrations on the Bcr-Abl carrying cells (Sun X et al Blood 97, 2008Sun X et al Blood 97, 2001). This data suggest that the tyrosine kinase inhibitors that target Bcr-Abl kinase may not be suitable for treating PV. We then tested the combination of AMN107 and AEE788 but did not observe synergistic effect at varying concentrations on reporter cells. The potent activity of AEE788 on JAK2 V617F suggests that AEE788 may act on unique target(s). We observed a decrease in Heat Shock Protein70 (HSP70) upon AEE788 treatment in HEL cells. Elevated levels of HSP70 are associated with drug resistance and poor prognosis (Creagh EM et al, Leukemia14: 1161, 2000). Down-regulation of HSP70 by anti-sense (Nylandsted et al, PNAS97:7871, 2000) and other interventions (Gaikwad A et al, Clin. Cancer Res 11: 1953Gaikwad A et al, Clin. Cancer Res 11: 2005) show the ability to overcome apoptotic resistance. Furthermore, AEE788 showed a specific inhibitory effect on native PV cells when we used in vitro expanded erythroid progenitors from PV patients. These treated PV expanded cells also showed down-regulation of HSP70. We conclude that AEE788 should be explored for the therapeutic trials of PV.

Description: Polycythemia vera (PV), the most common myeloproliferative disorder, arises due to somatic mutation(s) of a single hematopoietic stem cell leading to clonal hematopoiesis. A somatic JAK2 V617F point mutation is found in over 80% of PV patients; however, it is not clear if the JAK2 V617F is the disease initiating mutation, sincethere are PV JAK2 V617F negative patients who have monoclonal hematopoiesis and erythropoietin independent erythropoiesis;in individual PV families, there are PV subjects with and without the JAK2 V617F mutation; andanalysis of clonal PV populations reveals the presence of 50% mutated JAK2 cells (Nussenzweig’ abstract this mtg), suggesting a mixed population of cells with regard to JAK2 status.In order to search for possible PV contributing molecular defect(s), we studied microRNAs (miRNAs) in a homogeneous population of in vitro expanded erythroid progenitors. MiRNAs are non-coding, small RNAs that regulate gene expression at the posttranscriptional level by direct mRNA cleavage, by translational repression, or by mRNA decay mediated by deadenylation. MiRNAs play an important regulatory role in various biological processes including human hematopoiesis. In vitro expanded erythroid progenitors were obtained from peripheral blood mononuclear cells of 5 PV patients (JAK2 V617F heterozygotes) and from 2 healthy donor controls. The cells were cultured in an erythroid-expansion medium for 21 days resulting in 70–80% homogenous erythroid cell population of identical differentiation stage. Gene expression profiling of miRNAs (Thomson, Nature Methods, 1:1, 2004) was performed using a custom microarray (Combimatrix) with 326 miRNA probes. Data were normalized by the global median method. The miRNAs with expression ratios greater than 1.5 or less than 0.5 were considered to be abnormal. Comparative analyses of controls versus PV samples revealed up-regulated expression of miR-let7c/f, miR-16, miR-451, miR-21, miR-27a, miR-26b and miR-320 and down-regulation of miR-150, miR-339 and miR-346 in PV. In addition, miR-27a, miR-26b and miR-320 were expressed only in PV. The putative targets of these miRNAs were predicted by TargetScan prediction algorithm. Up-regulated miR-let-7, miR-16 and miR-26b may modulate cyclin D2, which has an important role in G1/S transition and can be a target in the JAK2/STAT5 pathway (Walz, JBC, 281:18177, 2006). One of the putative targets of up-regulated miR-27a is EDRF1 (erythroid terminal differentiation related factor1), a positive regulator of erythroid differentiation. The BCL-6 gene is predicted to be the target of miR-339 and miR-346, and its activation blocks cellular differentiation. MiR-16 is known to be down-regulated in CLL, where it targets anti-apoptotic BCL-2; in contrast, we show that miR-16 is up-regulated in PV erythroid cells. We identified differentially expressed miRNAs in PV which target genes involved in the JAK/STAT pathway or genes that are modulated by JAK2 downstream molecules. This study indicates that miRNA dysregulation may play an important role in erythropoietic differentiation and proliferation in PV. Expression analyses of these miRNAs in a larger set of PV samples, using quantitative Real-Time-PCR, are in progress. Further, earlier erythroid and pluripotent hematopoietic progenitors are also being analyzed.

Description: Juvenile myelomonocytic leukemia (JMML) is a rare mixed myelodysplastic/myeloproliferative neoplasm seen in early childhood. JMML in general is a poor prognosis disease, including risk of transformation to acute leukemic blast crisis in approximately 15% of patients. Like adult chronic myeloid leukemia (CML) the blast phase of JMML is typically myeloid, however B cell and T cell transformations have been reported. Interestingly, in CML, a minority of patients will have a small aberrant B-lymphoblast population that does not inevitably herald progression to B-lymphoblastic blast crisis, however this has not been reported in JMML. Here, we report a case of a child with JMML found to have an aberrant population of precursor B lymphoid blasts. An 11-month-old boy presented to our clinic with splenomegaly and complete blood count (CBC) findings of a low platelet count of 69 x103/UL, low absolute neutrophil count of 0.61 X103/UL and elevated monocytes (36.3%), with an absolute monocyte count of 3.2 x 103/UL. Bone marrow aspirate and biopsy were consistent with JMML with no abnormal blast population reported at the time. Karyotyping confirmed the presence of monosomy 7, with concurrent fluorescent in-situ hybridization (FISH) evaluation showing monosomy 7 in 74% of cells examined. Next generation sequencing on the aspirate revealed a KRAS p.G12A activating mutation at an allele frequency of 35%, further validating the diagnosis of JMML. The patient did not have a matched related or unrelated donor available, thus he was followed closely with serial physical exams, blood counts and bone marrow evaluations. His splenomegaly resolved, blood counts remained stable to improved without intervention, but repeat bone marrow 6 months after the diagnosis showed similar frequencies of monosomy 7 and mutant KRAS. With still no suitable donor available, we opted to pursue a trial of hypomethylating agent, 5-azacitidine that has shown promising results in recent reports. A bone marrow evaluation was done prior to starting azacitidine to establish a pre-therapy baseline. Surprisingly, we were halted by detecting a B lymphoid blast population (6%), with characteristic flow findings, including positive CD45 (dim), CD19, CD10, CD20 (partial), CD22 (dim), CD34, HLA-DR, CD52, CD99, CD58 (heterogeneous) and CD38. Additionally, a new partial CDKN2A deletion was detected by FISH in 10% of cells. These findings were concerning for an emerging precursor B-acute lymphoblastic leukemia, possibly driven by the new CDKN2A deletion. However, we flow sorted the aberrant B cell population from the marrow aspirate, which showed the partial CDKN2A deletion in only 1.25% of these cells, unlike monosomy 7, which was seen in 100% of the abnormal B cell population. A retrospective review of the flow data from the marrow 4 months prior revealed the same aberrant B cell population in a similar percentage (5%) that was originally attributed to hematogones with low CD45 expression. The stability of this population over the course of months was inconsistent with an evolving lymphoid blast crisis. We observed the patient closely, and his blood counts and clinical exam remained stable over the following weeks. A repeat bone marrow showed that the abnormal B-cell population had decreased slightly to 2.6% and the partial CDKN2A deletion was now seen in only 0.4% of the examined cells. Thereafter, treatment with azacitidine (5 daily doses-100mg/m2/dose) was initiated with an excellent response after one cycle, with monosomy 7 detected in only 12% of cells and KRAS allelic burden down to 4.3%. Additionally, the aberrant B cell population declined to 0.15% and partial CDKN2A deletion was not detected by FISH. While longer follow up and additional patients will be necessary to reach definitive conclusions, our case suggests that the immunophenotypic detection of a small, stable abnormal B-lymphoblast population in patients with JMML does not necessarily herald impending acute leukemia. Disclosures No relevant conflicts of interest to declare.

Description: Background: Compared to older children with ALL who achieve a minimal residual disease (MRD)-negative remission, infants with ALL who achieve an MRD-negative remission have a significantly higher rate of relapse. This suggests that, despite the achievement of MRD negativity, a very small amount of leukemia cells persist and contribute to disease relapse in infant ALL; LICs are likely to represent this persistent population of cells. Previously, we demonstrated that infant MLL-R ALL derive significantly more protection from co-culture with bone marrow (BM) stromal cells than non-MLL-R ALL. Therefore, protection by the BM microenvironment may contribute to LIC persistence in infant MLL-R ALL. We hypothesized that infant MLL-R ALL LICs have increased interactions with the BM microenvironment, through increased surface expression of adhesion molecule receptors. We also hypothesized that co-culture with normal BM stroma would protect LICs from spontaneous and chemotherapy-induced apoptosis, and AMD3100 (plerixafor) would decrease stromal protection through inhibition of CXCR4-CXCL12 signaling. Methods/Results: We analyzed 9 viably cryopreserved diagnostic samples collected from infants with MLL-R ALL (n=4 MLL-AF4, n=5 MLL-ENL). Using flow cytometry, we identified 2 phenotypically-defined LIC subpopulations, CD34+CD38+CD19+ and CD34-CD19+ (Aoki et al 2015), within the bulk leukemic blast population (CD45+). Consistent with previous findings, 34+38+19+ LICs were predominant in MLL-AF4 samples, while 34-19+ LICs were predominant in MLL-ENLsamples. Next, we measured surface expression of the adhesion molecule receptors CXCR4, CD49d (VLA-4), CXCR7, and CXCR3 (quantified as mean fluorescence intensity, MFI). Overall, surface expression of CXCR4, CD49d, CXCR7, and CXCR3 was higher in the 34+38+19+ LICs, compared to 34-19+ LICs (e.g., CXCR4 MFI 142 in 34+38+19+ vs. 76 in 34-19+, p=0.02). Next, samples were treated with dose ranges (0-30 µM) of AraC or etoposide (Etop) and cultured for 48 hours in 3 conditions: 1) off stroma, 2) on stroma, or 3) on stroma with the CXCR4 inhibitor AMD3100 (10 μM). Stromal cells were cultured from a healthy BM donor. In vehicle control-treated 34+38+19+ LICs, stromal co-culture led to increased surface expression of CD49d (avg 64% increase, p=0.03) and CXCR7 (avg 67% increase, p=0.02). Conversely, in vehicle control-treated 34-19+ LICs, stromal co-culture did not affect surface expression of the measured adhesion molecule receptors. In all 3 culture conditions, surface expression of CXCR4, CD49d, and CXCR7 was significantly higher in 34+38+19+ LICs compared to 34-19+ LICs (p≤0.01). We then measured apoptosis by flow cytometry and Annexin V binding. In the absence of chemotherapy, stromal co-culture significantly protected 34+38+19+ LICs from spontaneous apoptosis (p