ALBERT

Description: The C-Myb transcription factor is essential for hematopoiesis, including in the T-cell lineage. The C-Myb locus is a common site of retroviral insertional mutagenesis, however no recurrent genomic involvement has been reported in human malignancies. Here, we identified 2 types of genomic alterations involving the C-MYB locus at 6q23 in human T-cell acute leukemia (T-ALL). First, we found a reciprocal translocation, t(6;7)(q23;q34), that juxtaposed the TCRB and C-MYB loci (n = 6 cases). Second, a genome-wide copy-number analysis by array-based comparative genomic hybridization (array-CGH) identified short somatic duplications that include C-MYB (MYBdup, n = 13 cases of 84 T-ALL, 15%). Expression analysis, including allele-specific approaches, showed stronger C-MYB expression in the MYB-rearranged cases compared with other T-ALLs, and a dramatically skewed C-MYB allele expression in the TCRB-MYB cases, which suggests that a translocation-driven deregulated expression may overcome a cellular attempt to down-regulate C-MYB. Strikingly, profiling of the T-ALLs by clinical, genomic, and large-scale gene expression analyses shows that the TCRB-MYB translocation defines a new T-ALL subtype associated with a very young age for T-cell leukemia (median, 2.2 years) and with a proliferation/mitosis expression signature. By contrast, the MYBdup alteration was associated with the previously defined T-ALL subtypes.

Description: BACKGROUND: Conventional treatment options for patients with relapsed hematologic malignancies are both limited and highly toxic driving the pursuit of more tumor specific and less toxic therapies. RNA targeted oligonucleotides are potentially powerful drugs with the ability to silence genes required for malignant hematopoietic cell growth at the post transcriptional level. Studies from our laboratory have validated the c-myb proto-oncogene, which regulates important hematopoietic cell functions and is overexpressed in many hematologic malignancies, as a target for this technology. A prior Phase I trial using a 24 nucleotide phosphorothioated antisense oligodeoxynucleotide targeted to c-Myb mRNA (C-MYB AS ODN) did not identify a maximum tolerated dose (MTD) of the drug. Here we report initial results of a follow up Phase I dose escalation trial using C-MYB AS ODN at higher dose levels than previously studied in subjects with refractory hematologic malignancies. METHODS: C-MYB AS ODN is administered as a 7 day continuous infusion. 5 dose levels ranging from 3mg/kg/day to 12mg/kg/day are planned. Subjects are enrolled using an accelerated dose escalation scheme in which one subject is enrolled on each dose level (DL) with plans to revert to the standard 3+3 design in the event of significant attributable toxcity. C-MYB AS ODN concentrations are measured in peripheral blood (PB) and in mononuclear cells (MNC) by slot blotting at baseline, days 3 and 7 of infusion and two weeks after cessation of infusion. C-myb expression is assessed at these timepoints though QRT-PCR for c-myb RNA. Disease specific assessments of response are measured at predefined timepoints after therapy. RESULTS: 6 subjects, all with refractory acute myelogenous leukemia (AML) have enrolled to date. Escalation through the first 3 DLs occurred without any toxicities. At DL4 (10mg/kg/day) abnormalities have been noted in coagulation assays. The first subject enrolled on DL 4 developed a grade 3 prolongation of the activated partial thromboplastin time (PTT) attributable to drug which returned to normal within 48 hours of drug cessation. Factor levels, DIC parameters and reptilase time were normal. The PTT abnormality was consistent with a “lupus like” inhibitor effect (DRVVT was abnormal and the PTT corrected with the addition of phospholipid in two independent tests.) The 2nd subject treated at DL 4 developed a milder but similar PTT prolongation. The 3rd subject enrolled on DL 4 had a normal PTT throughout therapy. No subjects developed bleeding complications. Plasma and intracellular drug concentrations were dose related (320–640pg/l and 2–80 ng/5×10e6 cells respectively). Peak drug concentrations were found on Days 3–7. By 14 days after infusion, most ODN was cleared from plasma, but remained measurable in MNC at concentrations 30–50% of the maximum value detected. QRT-PCR for c-myb mRNA was performed in 3 subjects. Subject 1’s (DL 1) c-myb mRNA levels gradually decreased from baseline during infusion and nadired two weeks after cessation of infusion. Subjects 3 (DL 3) & 5 (DL 4) had a decrease in c-myb mRNA levels midway through infusion but c-myb RNA levels increased back to baseline by cessation of infusion. To date no subject has had a clinically important response to therapy. Accrual continues for DL 5. CONCLUSIONS: C-MYB AS ODN is detectable in plasma and MNCs of subjects during continuous drug infusion with a steady state reached by day 3 of infusion. Plasma drug levels were markedly reduced 14 days after cessation of infusion but MNC drug levels remained elevated. C-MYB AS ODN at DL 4 (10mg/kg/day) is associated with a PTT prolongation consistent with a “lupus inhibitor” like effect. While encouraging biological activity was identified optimal dose and delivery remain to be established before clinically significant effects can be reasonably expected.

Description: Biologic characterization of SB-559457 (SB), a nonpeptidyl hydrazone class of thrombopoietin receptor (Mpl) agonist, revealed toxicity toward human leukemia cells. Antiproliferative effects followed by significant, nonapoptotic, cell death within 72 hours occurred in 24 of 26 acute myeloid leukemia, 0 of 6 acute lymphoblastic leukemia, and 3 of 6 chronic myeloid leukemia patient samples exposed to SB, but not recombinant human thrombopoietin (rhTpo), in liquid suspension culture. Further investigation revealed increased phosphorylation of p70S6/S6 kinases in SB-, but not in rhTpo-, treated cells. Expression profiling of cells exposed to SB versus rhTpo revealed statistically significant, more than 2-fold changes in GAPDH and REDD1 gene expression, confirmed by quantitative reverse-transcribed polymerase chain reaction. These genes, induced in energy or hypoxia stressed cells, have been implicated in cell death pathways, and may provide important clues to the mechanism of SB-induced, leukemic cell death. These results suggest that nonpeptidyl, hydrazone class Mpl agonists may be clinically useful antileukemic agents by virtue of their combined thrombopoietic and antileukemic effects.

Description: Treatment of thrombocytopenia resulting from chemotherapy, radiation, or stem cell transplantation remains problematic. Hopes that TPO, the major regulator of megakaryocyte maturation, might prove clinically useful for this purpose have not been fulfilled, in part because of untoward reactions to the molecule. To avoid undesirable side effects, small molecule TpoR agonists are being developed. We have evaluated the biological effects of one such molecule, SB559457, a hydrazone compound, on hematopoietic cells. We first examined SB559457’s ability to stimulate proliferation of the recombinant human TPO (rhTPO) dependent cell line UT-7/TPO. We found a dose dependent augmentation of cell proliferation at doses between 1 and 10 μM, with maximal effects, compared to 100ng/ml of rhTPO at 10μM. We then evaluated SB559457’s growth promoting effects on normal human CD34+ cells. We again found growth stimulation to be dose dependent between 1 and 10 μM and also noted enhancement when SB559457 was combined with IL-3 and SCF. Specifically, incubation of CD34 cells in 10 μM SB559457 for 7 days lead to a 2 fold increase in cell number; incubation of cells with IL3 and SCF in addition to SB559457 lead to a 12 fold increase in cell number after 7 days. Both results were comparable to those seen with 100 ng/ml of rhTPO. Curiously, the addition of IL-6 to cultures containing IL-3 and SCF compromised the ability of SB559457, but not Tpo, to stimulate CD34+ cell growth. To determine if SB559457 stimulates megakaryocyte differentiation directly, CD34+ cells were incubated in cytokines with rhTPO or SB559457 for periods of 7–10 days, after which time cells in culture were examined for degree of polyploidization, and expression of megakaryocyte lineage markers CD41 and CD61. 14% of cells grown in SB559457 showed greater than 4N DNA content compared to 8% in rhTPO. These same cells expressed the megakaryocyte markers CD41 and CD61on 35% of cells, comparable to the results with rhTPO. Preliminary results demonstrate that incubation of CD34 cells in SB559457 maintained stem cells numbers, as assayed by NOD/SCID engraftment, for at least 4 days at a level comparable to the levels maintained by rhTPO. These results demonstrate that SB559457 is a bona fide Tpo R agonist that likely acts through activation of c-mpl, the receptor for TPO. These experiments provide a rationale for the evaluation of SB559457, or SB559457 congeners, in patients with thrombocytopenia.

Description: The c-myb proto-oncogene encodes an obligate hematopoietic cell transcription factor that contributes to lineage commitment, proliferation, and differentiation. Factors which regulate c-myb expression are of interest but remain incompletely defined. MicroRNAs (miRNAs) are being increasingly recognized as important regulators of cell development, and abnormalities in miRNA activity may also contribute to the pathogenesis of several hematologic malignancies. We speculated that miRNAs might also regulate c-Myb expression, a gene often aberrantly expressed in leukemia, lymphoma, and myeloma. Accordingly, we searched for potential miRNA binding sites in the 3′-UTR of the c-myb mRNA using the TargetScanS Target Database and identified 14 candidate miRNAs. Based on binding probability, three miRNAs (miR-15a, −107 and −150) were selected for further analysis using a luciferase reporter assay. 1191 bp of the human c-Myb 3′-UTR was subcloned downstream of the f-luc open reading frame to create the reporter construct (pBub1/Myb3U). This was co-transfected into HEK293T cell line with pRL-CMV (to normalize for transfection differences) and then either a control RNA oligonucleotide (ON) (miR-Control), miR-15a, miR-107, or miR-150 ONs. Relative luciferase activity of the pBub1/Myb3U construct was markedly diminished in cells co-transfected with miR-15a (74.1 ± 1.5%) or miR-107 (68.2 ± 5.3%) ONs, but only modestly with miR-150 ON (22.1 ± 4.2%). Functionality of miR-15a site was further tested by mutating the predicted miR-15a binding sites. This resulted in a 2 to 3 fold increase in luciferase activity, suggesting that miR-15a bound the predicated sites, and that they might be physiologically relevant. To test this possibility, miR-15a ONs were transfected into K562 human myeloid leukemia cells and the effects on c-Myb mRNA and MYB protein levels were determined. As expected with functional miRNA, c-myb mRNA levels did not change when compared to control treated cells, as measured by quantitative real-time PCR, but Myb protein levels were significantly decreased. Additionally, when analyzed by flow cytometry, miR-15a transfected cells were found to be arrested in G1 as might be expected in a Myb knockdown experiment. Conversely, when K562 cells were transfected with a methylphosphonate inhibitor (antisense) of miR-15a, endogenous c-myb expression increased. Moreover, exogenous expression in K562 cells of a c-Myb mRNA construct devoid of its 3′-UTR partially rescued the miR-15a induced cell cycle arrest. Interestingly, miR-15a levels were found to vary inversely with c-Myb mRNA expression levels in normal human CD34+ cells stimulated to develop along the erythroid, but not the myeloid lineage. These results suggested that miR-15a might play a specific, and potentially important role in regulating normal human erythropoiesis by modulating the expression of c-Myb, though we cannot exclude the possibility that additional miR-15a targets are also important. Finally, a siRNA mediated knockdown of c-Myb expression in K562 cells resulted in reduction of miR-15a expression, suggesting the possibility that Myb and miR-15a autoregulate their expression through a negative feed-back loop. We conclude that miR-15a regulates c-Myb expression in a physiologically significant, lineage specific manner in normal human hematopoietic cells. Since miR-15a localizes to ch13q14, a region often deleted in hematologic malignancies, we postulate that it might also play a role in leukemogenesis. This possibility is under active investigation in our laboratory.

Description: The abl tyrosine kinase inhibitor (TKI) imatinib mesylate has revolutionized treatment of Chronic Myelogenous Leukemia (CML). Nonetheless, for the small percentage of chronic phase patients in whom resistance to imatinib develops, and for patients in accelerated phase/blast crisis, disease management is problematic. Second generation TKI may address the resistance issue, but serious cardiotoxicity might be a concern for all TKIs. We are therefore investigating other candidates for rationally directed CML therapy. Herein we report that targeting tubulin with computationally designed small molecules may prove useful for the treatment of TKI resistant CML cells. Tubulin inhibitors were obtained from Locus Pharmaceuticals, Blue Bell, PA. They were designed using a method that computes virtual inhibitor molecule binding efficiency after integrating free energy calculations from all chemically possible combinations of molecule fragment poses. Molecules that perform well in in silico screens are then synthesized for biological testing. We evaluated 3 such molecules (LP-261, LOC-011294, and LOC-011423), and 2 control compounds (LOC-007708, LP-590), on K562 human leukemia cells, murine BaF3 cells expressing wild type bcr-abl, or the Y253F, T315I, E255K, H296P and M351T kinase domain mutations (gift from B. Druker, Portland, Oregon), and on consenting donors of normal, and CML, bone marrow cells. LP-261, LOC-011294, and LOC-011423 all had significant activity in K562 cells and BaF3 cells expressing wild type bcr-abl. Inhibition of cell growth in these lines was ~90% when employed at concentrations of 〉 100nM. More importantly in BaF3 cells expressing each of the mutant abl kinases, including T315I, growth inhibition was also ~90%. Moreover, LP-261, and LOC-011294 were also highly effective against primary cells obtained from patients with chronic phase and blast crisis CML. Treatment of primary CML cells with LP-261 resulted in 〉80% inhibition of proliferation in all five CML patient samples when compared to control cells. LOC-011294 inhibited cell proliferation by 〉80% in 3 out of 5 primary patient samples, and by ~50% in one other. Additional testing revealed that LP-261 was not a substrate for the p-glycoprotein multi-drug resistance porter, and that it is orally bioavailable. Neither LOC-007708, (a selective inhibitor of p38 kinase), nor LP-590 (an inhibitor of p38, Flt-3 and tie-2 kinases) inhibited proliferation of the cells expressing mutated bcr-abl. Since these kinases are not thought to influence bcr-abl driven cell growth, these results were expected. In contrast, MOLM14 cells, which overexpress Flt-3, were profoundly inhibited by LP-590. Disease specificity is suggested by the fact that none of these compounds had any effect on growth of acute lymphoid leukemia (ALL) patient samples. To be clinically useful, it was important to demonstrate that normal CD34+ were less sensitive to the growth inhibitory effects of these compounds. For this purpose, normal CD34+ cells were exposed to LP-261, LOC-011294, and LOC-011423 for 24 hours at concentrations shown to inhibit CML cell growth and then plated in methylcellulose with cytokines. Under these conditions, no significant inhibition of CFU-GM, CFU-E, or BFU-E in comparison to control cells was shown. These results suggests that rationally designed anti-tubulin small molecules, alone, or in combination with other active agents, may prove quite useful for treating kinase inhibitor resistant, as well as de novo, CML. This hypothesis may be tested in the near future as an IND has been filed for LP-261.

Description: Background JNJ-63709178 (CD123xCD3) is a promising new bispecific antibody currently being investigated for the treatment for acute myeloid leukemia (AML), a disorder of early hematopoietic progenitor cells characterized by the proliferation and accumulation of immature, clonal, myeloid cells. Constitutive overexpression of the α-subunit of the interleukin-3 receptor (ie, IL-3αR/CD123) is a hallmark of the early AML progenitor cell population (ie, leukemic stem cells [LSC]), as well as leukemic blasts. CD123 is unique to the IL-3 receptor and is responsible for the high specificity and low affinity binding of IL-3. Under physiological conditions, activation of the IL-3 receptor induces proliferative, anti-apoptotic, and differentiating signals. JNJ-63709178, a humanized IgG4 bispecific DuoBody® antibody with high affinity anti-CD123 and -CD3 arms, recognizes CD123 on myeloid cells and CD3 on T cells, leading to T-cell activation, with subsequent lysis of CD123+ AML blasts. JNJ-63709178 induces potent tumor cell killing in vitro, ex vivo, and in murine AML models, and is currently being evaluated in first-in-human clinical trials. Although CD123 is distinctively expressed on leukemic cells, some populations of normal myeloid cells have been reported to express CD123. The goals of this study were to determine CD123 receptor density on various leukocyte populations in normal human peripheral blood and evaluate the effects of JNJ-63709178 on those cells to identify potential pharmacodynamic markers. Methods Peripheral blood samples from 6 normal human donors were analyzed ex vivo for CD123 expression on various leukocyte populations to determine receptor density. Receptor density on cells was quantified using Quantibrite PE enumeration kit. Subsequently, blood samples were treated with escalating concentrations of JNJ-63709178 (CD123xCD3) or the control compound CNTO 7008 (nullxCD3) DuoBody® antibodies and incubated at 37°C for 48 hours. After incubation, blood samples were stained and processed for analysis of T-cell activation (CD25 expression) and population persistence or depletion (frequencies within leukocytes). In the myeloid compartment, basophils, myeloid dendritic cells (mDCs), monocytes, neutrophils, and eosinophils were analyzed. In the lymphoid compartment, plasmacytoid dendritic cells (pDCs), B cells (naïve and memory), T cells (helper and cytotoxic), and natural killer (NK) cells (CD56bright and CD56dim) were analyzed. Results In normal human peripheral blood samples, CD123 expression was highest on pDCs and basophils (above 10,000 receptors per cell). Intermediate expression (300-3,000 receptors per cell) was observed on mDCs, monocytes, eosinophils, and naïve B cells. Low expression (10-200 receptors per cell) was observed on neutrophils, memory B cells, T cells, and NK cells. These data are in agreement with previous studies (Busfield et al. ASH 2013 abs3598). Following incubation with JNJ-63709178, potent activation of CD4+ and CD8+ T cells was observed at sub-nanomolar levels, with EC50values of 0.07 nM and 0.1 nM, respectively. Higher CD123 expression and T-cell activation correlated strongly with profound depletion of basophils and pDCs, as well as partial depletion of eosinophils and monocytes. Consistent with low CD123 expression, T cells and NK cells did not show substantial dose-dependent depletion. In contrast to CD123 expression levels, B cells and neutrophils showed partial depletion, despite low CD123 expression, while mDCs did not show dose-dependent depletion, despite higher CD123 levels. The activation of T cells and cytotoxic effect on CD123-positive cells was observed only with JNJ-63709178. The control compound CNTO 7008 (nullxCD3) had no effect on T-cell activation or any of the investigated cell populations. Conclusions These findings highlight the potential pharmacodynamic markers of JNJ-63709178 (CD123xCD3) bispecific treatment and provide a better understanding of its mechanism of action and a rationale for receptor expression monitoring during ongoing clinical trials. Disclosures Babich: Janssen: Employment. Li:Janssen: Employment. Gaudet:Janssen Pharmaceuticals R&D: Employment, Other: Stock options, Patents & Royalties: pending, not yet issued. Huang:Janssen Research & Development, LLC: Employment, Other: I am an employee of Janssen and a stock owner . Sasser:Johnson & Johnson: Equity Ownership; Janssen Pharmaceuticals R&D: Employment. Salvati:Janssen Pharmaceuticals R&D: Employment, Other: stock options, Patents & Royalties: patent. Kalota:Janssen Pharmaceuticals R&D: Employment, Other: stock.

Description: The c-myb proto-oncogene has been implicated in leukemogenesis, but possible mechanisms remain ill defined. To gain further insight to this process, we used transcript profiling in K562 cells expressing a dominant-negative Myb (MERT) protein. A total of 105 potential Myb gene targets were identified. Neuromedin U (NmU), a peptide affecting calcium transport, underwent the greatest expression change (∼ 5-fold decrease). To verify a linkage between c-myb and NmU, their mRNA levels were quantitated using real-time polymerase chain reaction in primary acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL), as well as normal hematopoietic cells. We found that c-myb was elevated in AML and ALL samples, but NmU expression was increased only in AML cells. Significantly, only AML cells expressed the cognate receptor of NmU, NMU1R, suggesting the presence of a novel autocrine loop. We examined this possibility in detail. Exogenous NmU “rescued” growth suppression in K562-MERT cells and stimulated the growth of primary AML cells. Short interfering RNA “knockdown” of NmU in K562 cells arrested cell growth. Exposing Indo-1–labeled K562 cells to NmU induced an intracellular Ca++ flux consistent with engagement of the NMU1R. Combined, these results suggest that NmU expression is related to Myb and that the NmU/NMU1R axis constitutes a previously unknown growth-promoting autocrine loop in myeloid leukemia cells.