ALBERT

Description: Formative research suggests that a human embryonic stem cell-specific alternative splicing gene regulatory network, which is repressed by Muscleblind-like (MBNL) RNA binding proteins, is involved in cell reprogramming. In this study, RNA sequencing, splice isoform-specific quantitative RT-PCR, lentiviral transduction, and in vivo humanized mouse model studies demonstrated that malignant reprogramming of progenitors into self-renewing blast crisis chronic myeloid leukemia stem cells (BC LSCs) was partially driven by decreased MBNL3. Lentiviral knockdown of MBNL3 resulted in reversion to an embryonic alternative splice isoform program typified by overexpression of CD44 transcript variant 3, containing variant exons 8–10, and BC LSC proliferation. Although isoform-specific lentiviral CD44v3 overexpression enhanced chronic phase chronic myeloid leukemia (CML) progenitor replating capacity, lentiviral shRNA knockdown abrogated these effects. Combined treatment with a humanized pan-CD44 monoclonal antibody and a breakpoint cluster region - ABL proto-oncogene 1, nonreceptor tyrosine kinase (BCR-ABL1) antagonist inhibited LSC maintenance in a niche-dependent manner. In summary, MBNL3 down-regulation–related reversion to an embryonic alternative splicing program, typified by CD44v3 overexpression, represents a previously unidentified mechanism governing malignant progenitor reprogramming in malignant microenvironments and provides a pivotal opportunity for selective BC LSC detection and therapeutic elimination.

Description: Background: Cirmtuzumab is a first-in-class humanized mAb specific for ROR1, an oncoembryonic antigen found on CLL and cancer stem cells of various cancers, but not on normal post-partum tissues. Work has defined that ROR1 serves as a receptor for Wnt5a, which induces non-canonical Wnt-signaling that promotes planar-cell-polarity, migration, stem-cell renewal, and proliferation. Recent studies demonstrated that Wnt5a binds to ROR1 and induces activation of RhoA and Rac1, promoting leukemia-cell migration and proliferation, respectively. By binding a functional epitope in the extracellular domain of ROR1, cirmtuzumab can block ROR1-dependent, non-canonical Wnt5a-signaling. Methods: We conducted a phase 1 trial of cirmtuzumab in patients (pts) with progressive, relapsed/refractory CLL who were not amenable to approved therapies. The primary aims of the study were to evaluate the safety and tolerability of cirmtuzumab and determine the maximum tolerated dose and/or recommended phase 2 dose. Secondary endpoints included evaluation of antibody pharmacokinetics (PK) and pharmacodynamics (PD). To address these endpoints, pts received only 4 biweekly infusions of antibody at doses ranging from 15 mcg/kg to 16 mg/kg in a standard 3+3 pt-per-cohort schema. Results: As of a planned analysis, 20 pts have received cirmtuzumab. Pts tolerated cirmtuzumab extremely well without any noted drug-related severe adverse events or dose-limiting toxicities. Anemia (7 pts), thrombocytopenia (4 pts), and neutropenia (3 pts) were the most common AEs, were primarily grade 1, and were likely due to late-stage-disease-related cytopenias. We developed an ELISA assay to measure serum levels of cirmtuzumab capable of binding to the targeted epitope of ROR1. Peak concentrations were detected within one hour after the completion of each infusion. The serum concentrations of active cirmtuzumab increased with each subsequent infusion. We detected significant levels of cirmtuzumab for at least eight weeks following the last infusion and determined that it has half-life of 〉24 days (d). Twenty-four hours following the 1st infusion, the leukemic cells of pts treated at doses 〉/= 2 mg/kg had inactivation of RhoA and Rac1, which were each observed to be activated in all cases prior to therapy. Loss of GTPase activation also was observed for CLL cells sampled at later time points. Clinically, we observed that most pts had an initial increase in the ALC without evidence of disease progression, similar to what is observed with targeted therapies that inhibit B-cell-receptor/chemokine signaling. Such a redistributive lymphocytosis (defined as an increase in the ALC of 10% or more) was observed in 5 of 9 pts who received doses /=2mg/kg. The lymphocytosis typically peaked 24 hours after dosing (median 36% increase, range 10-76%), with a subsequent reduction to at or below baseline levels. Most pts had stable disease when assessed at the completion of treatment. Of evaluable pts, 12 had stable disease and 2 had progressive disease, both of whom received

Description: Background Acute myelogenous leukemia (AML) is a highly lethal disease with only 20% of 5 years survival. Most AML patients experience relapse usually leading to death. Progression to therapy resistant AML is driven by leukemia stem cells (LSC) harboring enhanced survival, dormancy and self-renewal capacity in supportive niches. Growing evidence indicates that in the development of myeloid malignancies deregulation of stem cells activity is as important as deregulation of the BM microenvironment. Sonic hedgehog (SHH) pathway molecules in general and SMO in particular are well known to maintain the growth of cancer cells. However, it’s role in leukemia niche-stem cell interaction is not well defined. Therefore, here we investigate: 1/ how the small molecule, SMO antagonist, PF-0449913 impacts the AML BM microenvironment and 2/ how in turn, changes in the activity of AML BM niche cells contribute to its remodeling into HSC supportive niche. Methods CD34- cell from bone marrow patients undergoing hip replacement surgery (normal BM) as well as AML bone marrow were utilized for the development of the primary human stromal monolayers. We investigated stromal cultures of primary normal BM (NBM, n=6) and AML (n=6). As a control human normal bone marrow stromal cell line HS-5 was used. Then human CD34+ cells were selected from AML primary samples (n=6). As a normal control, CD34+ cells from cord blood (CB, n=5), or NBM (n=5) were utilized for the co-culture experiments for up to 9 weeks and then plated in survival and self-renewal assays. PF-0449913 was added to stromal co-cultures or, as a pre-treatment, directly to stromal monolayers. Results In order to examine the role of SMO regulation in LSC generation and maintenance, primary AML (n=6) or cord blood (n=3) CD34+ cells were co-cultures with normal BM (n=3) or AML (n=3) stroma in the presence of novel small molecule inhibitor (PF-0449913) for 2 weeks. It significantly reduced LSC survival and self-renewal, while spared HSC. These effects were not observed in the absence of stroma. Importantly, pre-treatment of the AML stroma with PF-0449913 for 1 week prior- to co-culture also resulted in LSC’s inhibition of survival and self-renewal. AML- and normal BM-derived stroma differ in their ability to support HSC and LSC: LSC (n=6) were capable to self-renew after 9 weeks of co-culture with both normal and AML stroma, while cord blood (n=4) as well as normal BM (n=5) HSC lost their self-renewal potential after only 2 weeks of co-culture with the AML stroma. PF-0449913 induced changes in AML stroma. which was previously unsupportive to HSC survival, it became compliant : self-renewal of HSC increased more than 3 times. It reversed also the inhibitory effect of AML stroma-derived conditioned media (CM). Pre-treatment of the HS-5 cells with CM from AML stroma for 4 weeks prior to co-culture experiments led to significant inhibition of the cord blood (n=3) and NBM (n=3) HSC survival and self-renewal. However, in CM obtained from AML stroma treated with PF-0449913 that effect was reversed. Combined treatment of AML cells (n=6) with PF-0449913 and de-methylating agent Vidaza (5-azacytodine) or CXCR4 antagonist AMD3100 resulted in even greater inhibition of LSC survival and self-renewal. However, Vidaza did not mediate changes in AML stroma. Conclusions Together these data indicate that, while both conditioned media and co-culture with AML stroma impaired HSC survival and self-renewal, co-culture conditions resulted in a greater reduction in survival and self-renewal capacity, implicating that cell-cell contact or unstable secreted factors exacerbate the effects. That suggests that microenvironmental cues play a key role in regulating normal HSC versus LSC survival and maintenance, and leukemic stroma exibit severely compromised ability to maintain normal HSCs, but effectively support LSCs. PF-0449913treatment of AML induces not only eradication of LSC, but also mediate changes in AML stroma, supporting HSC generation and maintenance. Simultaneous targeting of both AML niche and LSC could represent a novel avenue for treatment of AML patients. Disclosures No relevant conflicts of interest to declare.

Description: Background: NS-018 is an oral, selective, small molecule inhibitor of Janus kinase 2 (JAK2). OBJECTIVE: The purpose of this study is to determine the safety, tolerability and efficacy of orally administered NS-018 in patients with PMF, post-PV MF, or post-ET MF. METHODS: This multicenter, Phase 1/2, 3+3 dose-escalation study of NS-018 enrolled patients with IPSS intermediate-1, intermediate-2, or high risk PMF, post-PV MF, or post-ET MF. The study drug, NS-018, was given orally either daily (QD) or twice daily (BID) in 28-day cycles. In Phase 1, changes in spleen size were assessed by manual palpation, quality of life with the Myelofibrosis Symptom Assessment Form (MF-SAF), and responses were assessed according to the IWG-MRT/ELN consensus criteria. Bone marrow fibrosis (BMF) was evaluated by biopsy, according to WHO criteria. Changes in grade of BM fibrosis from baseline were categorized as improvement, stabilization, or worsening. The Phase 1 portion of the study has been completed and data are presented here. RESULTS: In this Phase 1 study, 48 patients were enrolled across 10 dosing cohorts (75-400 mg QD/100-400 mg BID). Characteristics: 37 PMF, 5 post-PV MF, 6 post-ET MF with a median age (range) 69.5 yrs (38-83); M/F:29/19; 35 JAK2V617F+, and 23 previously treated with a different JAK2 inhibitor. At 400 mg QD/BID, NS-018 dosing was associated with drug-related neurologic adverse events (AEs) including dizziness, peripheral neuropathy, headache, disturbance in attention, vertigo, dysesthesia, paresthesia, aphasia, and nervous system disorder (not otherwise specified). The 300 mg QD dose was better tolerated than 250 mg BID or 300 mg BID, with fewer neurologic AEs, and was selected as the recommended phase 2 dose (RP2D). For the 48 phase 1 patients, reductions in MF-SAF score were observed for all symptoms after 3 cycles, including for patients with prior JAK2 inhibitor treatment. In all dose cohorts, ≥ 50% patients achieved ≥ 50% score reduction from baseline in night sweats, pruritus and bone pain after 1 cycle. Moreover, ≥ 50% patients achieved ≥ 50% score reduction from baseline in abdominal pain and inactivity after 3 cycles. In patients who had received prior JAK2 inhibitor, ≥ 50% patients achieved ≥ 50% score reduction from baseline in filling up quickly, inactivity, night sweats and quality of life after 3 cycles. Among 36 patients with baseline splenomegaly ≥ 5 cm and treatment for ≥ 1 cycle, 20 (56%) showed ≥ 50% reduction in spleen size (confirmed for ≥ 8 weeks in 16 patients), including 9/19 (47%) patients with prior JAK2 treatment. According to IWG criteria, 14/36 (39%) patients showed splenic clinical improvement (CI) for ≥ 8 weeks, 4 had hemoglobin CI, and 1 had platelet CI. After 3 cycles of NS-018 treatment, 11/31 (37%) evaluable patients had a reduction in bone marrow fibrosis(BMF) by ≥ 1 grade. At any time during NS-018 treatment, 17/31 (55%) evaluable patients had a reduction in BMF by ≥ 1 grade. Overall, 23% (7/31) patients maintained improvement in BMF for ≥ 12 weeks. Reduction in BMF after 3 cycles was correlated with reduction in splenomegaly and hemoglobin responses. To date, 8 patients with prior JAK inhibitor treatment have been enrolled into the phase 2 portion of the trial. Updated Phase 2 data will presented at the meeting. CONCLUSIONS: The RP2D dose of NS-018 was 300 mg QD. This dose provided an acceptable safety and tolerability profile, and reduction in symptomatic splenomegaly. Phase 2 is ongoing and includes patients previously treated with other JAK2 inhibitors. Disclosures Talpaz: ARIAD: Research Funding; Bristol-Myers Squibb: Research Funding; Sanofi Aventis: Research Funding; Pfizer: Research Funding; Incyte: Research Funding. Ritchie:Incyte: Speakers Bureau; Celgene: Speakers Bureau. Odenike:Spectrum Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Algeta Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; Suneisis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; CTI/Baxter: Honoraria, Membership on an entity's Board of Directors or advisory committees; Geron: Research Funding. Jamieson:GlaxoSmithKline: Research Funding; Johnson & Johnson: Research Funding. Stein:Incyte Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees. Mesa:Novartis Pharmaceuticals Corporation: Consultancy; Pfizer: Research Funding; CTI Biopharma: Research Funding; NS Pharma: Research Funding; Gilead: Research Funding; Incyte Corporation: Research Funding; Promedior: Research Funding; Genentech: Research Funding.

Description: The translation of genomic discoveries to the clinic is the cornerstone of precision medicine. However, incorporating next generation sequencing (NGS) of hematologic malignancies into clinical management remains limited. We describe 235 such patients who underwent integrated NGS profiling (406 genes) and analyze the alterations and their potential actionability. Overall, 227 patients (96.5%) had adequate tissue. Most common diagnoses included myelodysplastic syndrome (22.9%), chronic lymphocytic leukemia (17.2%), non-Hodgkin lymphoma (13.2%), acute myeloid leukemia (11%), myeloproliferative neoplasm (9.2%), acute lymphoblastic leukemia (8.8%) and multiple myeloma (7.5%). Most patients (N = 197/227 (87%)) harbored ≥1 genomic alteration(s); 170/227 (75%), ≥1 potentially actionable alteration(s) targetable by an FDA-approved (mostly off-label) or an investigational agent. (Table 1) Altogether, 551 distinct alterations were seen, most commonly involving TP53 (9.3% of patients), TET2 (4.2%), DNMT3A (3.3%), ASXL1 (2.5%), MLL (2.5%), and KRAS (2.2%). (Figure 1 and Figure 2) The median tumor mutational burden (TMB) was low (1.7 alterations/megabase); 12% of patients had intermediate or high TMB (higher TMB correlates with favorable response to anti-PD1/PDL1 inhibition in solid tumors). In conclusion, 96.5% of patients with hematologic malignancies have adequate tissue for comprehensive genomic profiling. Most patients had unique molecular signatures, and 75% had alterations that may be pharmacologically tractable with gene- or immune-targeted agents. Disclosures No relevant conflicts of interest to declare.

Description: Gene expression profiles (GEPs) were obtained from marrow hematopoietic precursor cells (HPC)(CD34+ cells) from 30 myelodysplastic syndrome (MDS) patients: RARS 2, RA 15, RAEB 9, RAEBT 4; IPSS Low 11, Int-1 10, Int-2 5, High 4, and 6 Normal individuals. Fluorescently labeled cDNA was prepared from CD34+ cells (〉90% purity), isolated by immunomagnetic column separation, after reverse transcription of high fidelity PCR-amplified poly(A) RNA (aRNA). The Cy-conjugated nucleotides for aRNA were hybridized to 40,000 gene chip microarrays obtained from the Stanford Functional Genomics Microarray Facility. aRNA from pooled normal CD34+ marrow cells was used as a Reference standard. High resolution scans were obtained to compile a dataset for each microarray, through files submitted to the Stanford Microarray Database. Dendrograms generated by unsupervised hierarchical gene clustering indicated major differences of GEP between Normal and MDS patients. Significance Analysis for Microarray (SAM) yielded 2327 genes significantly differentially expressed by MDS vs Normal: 2269 genes overexpressed, 58 underexpressed, with a false positive rate of ~10%. Prediction Analysis of Microarray (PAM) distinctly separated the MDS and Normal patients, requiring a minimum of 31 genes (which were also SAM significant). Class analysis by PAM correctly predicted 29 of the 30 to be MDS and 5 of the 6 to be Normal. Four disparate differential GEP regions in the dendrograms, comprising predominantly genes of differing functional categories provided signatures associated with differing MDS clinical subgroups. Nine of 10 patients with poor clinical outcomes were associated with a differing GEP signature than that which occurred in 14 of 20 patients with relatively good outcomes. Compared to the remainder of MDS patients, those with 5q- syndrome (n=5) had a differing GEP signature, with under-expression of 1018 genes, 11 of which were within the 5q31–32 CDS. Two of these genes (antioxidant protein1 and interferon regulatory factor1) have previously been proffered as candidate genes for this syndrome. Analysis of FACS-sorted highly purified marrow HPC subsets: CD34+38+ (late) and CD34+38- (early HPCs), indicated these ratios to be 4.3±2.1 (n=2) for MDS and 3.2±1.2 (n=12) for Normals. These findings suggest that the differing GEPs between the MDS and Normal CD34+ cells were not due to major differences in their proportions of CD38 cell subsets. SAM and PAM significant differential GEPs were noted between these cell subsets (also differing between MDS and Normal), indicating alteration of gene expression during differentiation. Wnt1 and β-catenin1 (genes involved in cell self-renewal) were over-expressed in both MDS CD38- and CD38+ cells compared to Normal. These data demonstrate: (1) molecular differences between MDS and Normal HPCs and within HPC subsets; (2) GEP signatures characterizing MDS patients with differing cytogenetic abnormalities (eg, 5q-) and clinical outcomes; (3) molecular criteria refining the prognostic categorization of MDS; and (4) gene expression data aiding characterization of the heterogeneous nature of this spectrum of diseases.

Description: Recent evidence suggests that a rare population of self-renewing cancer stem cells (CSC) is responsible for cancer progression and therapeutic resistance. Chronic myeloid leukemia (CML) represents an important paradigm for understanding the genetic and epigenetic events involved in CSC production. CML progresses from a chronic phase in hematopoietic stem cells (HSC) that harbor the BCR-ABL translocation, to blast crisis, characterized by aberrant activation of β-catenin within granulocyte-macrophage progenitors (GMP). Here we show that blast crisis CML myeloid progenitors can serially transplant leukemia in immunocompromised mice, and thus are enriched for leukemia stem cells (LSC). Genomic DNA and cDNA sequencing analysis of Wnt/β-catenin pathway regulatory genes including APC, GSK3β, axin 1, β-catenin, LEF-1, cyclin D1 and c-myc in 49 normal and 51 CML HSC, GMP and lineage-positive populations revealed that in 57% of blast crisis samples a novel in-frame splice deletion of the GSK3β kinase domain occurred in blast crisis CML GMP, but not blasts found in blast crisis or normal progenitors. CML progenitors with misspliced GSK3β have enhanced β-catenin expression as well as serial engraftment potential. We propose that chronic phase CML is initiated by BCR-ABL expression in an HSC clone but that progression to blast crisis may include missplicing of GSK3β in GMP LSC, enabling unphosphorylated β-catenin to participate in LSC self-renewal. Missplicing of GSK3β represents an unique mechanism for the emergence of blast crisis CML LSC, is a potential diagnostic indicator of disease progression and represents a novel therapeutic target in blast crisis CML. As the first description of GSK3β dysregulation in cancer, this research provides a focal point for investigating whether GSK3β missplicing also triggers β-catenin activation in other CSC.

Description: Chronic myelomonocytic leukemia (CMML) is a unique myeloproliferative disease characterized by marrow dysplasia and an increase in monocytes. The median survival of patients with CMML is short, in part, because CMML is frequently resistant to therapy. In order to provide more effective CMML targeted therapies, a better understanding of the basic biology of CMML progenitors is required. We used FACS analysis and recently identified cell surface markers to identify phenotypic and functional differences between normal and CMML (n=14) bone marrow hematopoietic stem cells and myeloid progenitors. CMML marrow was typified by a reduction in CD34+CD38−CD90+(Thy1)Lin− hematopoietic stem cells and an expansion of CD34+CD38−CD90−Flk2+Lin- cells relative to normal bone marrow. In addition, there was a two-fold expansion in common myeloid progenitors (CMPs) and a corresponding decrease in megakaryocyte-erythroid progenitors (MEPs) suggesting that there was a skew in differentiation toward the myeloid lineage. In contrast to normal bone marrow derived CMPs, CMML CMPs gave rise to myeloid but not erythroid colonies. Moreover, real time quantitative RT-PCR analysis of highly purified FACS-sorted CMML CMPs demonstrated increased expression of two key regulators of myelomonocytic differentiation, PU.1 and c-jun, compared with normal bone marrow. A more detailed understanding of the basic biology of CMML myeloid progenitors and the genes that work in concert to expand them may aid in identifying novel molecular targets for CMML therapy.