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Phenotypic characterization of T cells in myelofibrosis is intriguing because of increased inflammation, markedly elevated pro-inflammatory cytokines, and altered distribution of T-cell subsets. Constitutive activation of Janus kinase-2 (JAK2) in the majority of patients with myelofibrosis contributes to the expression of the programmed cell death protein-1 (PD1) and T-cell exhaustion. We wondered whether T-cell activation affects treatment outcome of patients with myelofibrosis and sought to determine whether the JAK1/2 inhibitor ruxolitinib affects the activation of T-cell subsets. T cells from 47 myelofibrosis patients were analyzed and the percentages of either helper (CD4+) or cytotoxic (CD8+) naïve, central memory, effector memory, or effector T cells; and fractions of PD1-expressing cells in each subset were assessed. Higher numbers of T cells co-expressing CD4/PD1 and CD8/PD1 were found in myelofibrosis patients than in healthy controls (n=28), and the T cells were significantly skewed toward an effector phenotype in both CD4+ and CD8+ subsets, consistent with a shift from a quiescent to an activated state. Over the course of ruxolitinib treatment, the distribution of aberrant T-cell subsets significantly reversed towards resting cell phenotypes. CD4+ and CD8+ subsets at baseline correlated with monocyte and platelet counts, and their PD1+ fractions correlated with leukocyte counts and spleen size. Low numbers of PD1+/CD4+ and PD1+/CD8+ cells were associated with complete resolution of palpable splenomegaly and improved survival rate, suggesting that low levels of exhausted T cells confer a favorable response to ruxolitinib treatment.

The promising activity of BET protein inhibitors (BETi’s) is compromised by adaptive or innate resistance in acute myeloid leukemia (AML). Here, modeling of BETi-persister/resistance (BETi-P/R) in human postmyeloproliferative neoplasm (post-MPN) secondary AML (sAML) cells demonstrated accessible and active chromatin in specific superenhancers/enhancers, which was associated with increased levels of nuclear β-catenin, TCF7L2, JMJD6, and c-Myc in BETi-P/R sAML cells. Following BETi treatment, c-Myc levels were rapidly restored in BETi-P/R sAML cells. CRISPR/Cas9-mediated knockout of TCF7L2 or JMJD6 reversed BETi-P/R, whereas ectopic overexpression conferred BETi-P/R in sAML cells, confirming the mechanistic role of the β-catenin–TCF7L2–JMJD6–c-Myc axis in BETi resistance. Patient-derived, post-MPN, CD34+ sAML blasts exhibiting relative resistance to BETi, as compared with sensitive sAML blasts, displayed higher messenger RNA and protein expression of TCF7L2, JMJD6, and c-Myc and following BETi washout exhibited rapid restoration of c-Myc and JMJD6. CRISPR/Cas9 knockout of TCF7L2 and JMJD6 depleted their levels, inducing loss of viability of the sAML blasts. Disruption of colocalization of nuclear β-catenin with TBL1 and TCF7L2 by the small-molecule inhibitor BC2059 combined with depletion of BRD4 by BET proteolysis-targeting chimera reduced c-Myc levels and exerted synergistic lethality in BETi-P/R sAML cells. This combination also reduced leukemia burden and improved survival of mice engrafted with BETi-P/R sAML cells or patient-derived AML blasts innately resistant to BETi. Therefore, multitargeted disruption of the β-catenin–TCF7L2–JMJD6–c-Myc axis overcomes adaptive and innate BETi resistance, exhibiting preclinical efficacy against human post-MPN sAML cells.

Bone marrow (BM) sclerosis is commonly found in patients with late-stage myelofibrosis (MF). Because osteoclasts (OCs) and osteoblasts play a key role in bone remodeling, and MF monocytes, the OC precursors, are derived from the neoplastic clone, we wondered whether decreased OC numbers or impairment in their osteolytic function affects the development of osteosclerosis. Analysis of BM biopsies from 50 MF patients showed increased numbers of multinucleated tartrate-resistant acid phosphatase (TRAP)/cathepsin K+ OCs expressing phosphorylated Janus kinase 2 (JAK2). Randomly microdissected TRAP+ OCs from 16 MF patients harbored JAK2 or calreticulin (CALR) mutations, confirming MF OCs are clonal. To study OC function, CD14+ monocytes from MF patients and healthy individuals were cultured and differentiated into OCs. Unlike normal OCs, MF OCs appeared small and round, with few protrusions, and carried the mutations and chromosomal abnormalities of neoplastic clones. In addition, MF OCs lacked F-actin–rich ring-like structures and had fewer nuclei and reduced colocalization signals, compatible with decreased fusion events, and their mineral resorption capacity was significantly reduced, indicating impaired osteolytic function. Taken together, our data suggest that, although the numbers of MF OCs are increased, their impaired osteolytic activity distorts bone remodeling and contributes to the induction of osteosclerosis.

There is an unmet need to overcome nongenetic therapy-resistance to improve outcomes in AML, especially post-myeloproliferative neoplasm (MPN) secondary (s) AML. Studies presented describe effects of genetic knockout, degradation or small molecule targeted-inhibition of GFI1/LSD1 on active enhancers, altering gene-expressions and inducing differentiation and lethality in AML and (MPN) sAML cells. A protein domain-focused CRISPR screen in LSD1 (KDM1A) inhibitor (i) treated AML cells, identified BRD4, MOZ, HDAC3 and DOT1L among the codependencies. Our findings demonstrate that co-targeting LSD1 and one of these co-dependencies exerted synergistic in vitro lethality in AML and post-MPN sAML cells. Co-treatment with LSD1i and the JAKi ruxolitinib was also synergistically lethal against post-MPN sAML cells. LSD1i pre-treatment induced GFI1, PU.1 and CEBPα but depleted c-Myc, overcoming nongenetic resistance to ruxolitinib, or to BETi in post-MPN sAML cells. Co-treatment with LSD1i and BETi or ruxolitinib exerted superior in vivo efficacy against post-MPN sAML cells. These findings highlight LSD1i-based combinations that merit testing for clinical efficacy, especially to overcome nongenetic therapy-resistance in AML and post-MPN sAML.

The glioma associated oncogene-1 (GLI1), a downstream effector of the embryonic Hedgehog pathway, was detected in chronic lymphocytic leukemia (CLL), but not normal adult cells. GLI1-activating mutations were identified in 10% of patients with CLL. However, what induces GLI1 expression in GLI1-unmutated CLL cells is unknown. Because signal transducer and activator of transcription 3 (STAT3) is constitutively activated in CLL cells, and sequence analysis detected putative STAT3-binding sites in the GLI1 gene promoter, we hypothesized that STAT3 induces the expression of GLI1. Western immunoblotting detected GLI1 in CLL cells from 7 of 7 patients, flow cytometry analysis confirmed that CD19+/CD5+ CLL cells co-express GLI1, and confocal microscopy showed co-localization of GLI1 and phosphorylated STAT3. Chromatin immunoprecipitation showed that STAT3 protein co-immunoprecipitated GLI1, as well as other STAT3-regulated genes. Transfection of CLL cells with STAT3-shRNA induced a mark decrease in GLI1 levels, suggesting that STAT3 binds to and induces the expression of GLI1 in CLL cells. An electromobility shift assay confirmed that STAT3 binds, and a luciferase assay showed that STAT3 activates the GLI1 gene. Transfection with GLI1-siRNA significantly increased the spontaneous apoptosis rate of CLL cells, suggesting that GLI1 inhibitors might provide therapeutic benefit to patients with CLL.

The glioma associated oncogene-1 (GLI1), a downstream effector of the embryonic Hedgehog pathway, was detected in chronic lymphocytic leukemia (CLL), but not normal adult cells. GLI1 activating mutations were identified in 10% of patients with CLL. However, what induces GLI1 expression in GLI1-unmutated CLL cells is unknown. Because signal transducer and activator of transcription 3 (STAT3) is constitutively activated in CLL cells and sequence analysis detected putative STAT3-binding sites in the GLI1 gene promoter, we hypothesized that STAT3 induces the expression of GLI1. Western immunoblotting detected GLI1 in CLL cells from 7 of 7 patients, flow cytometry analysis confirmed that CD19+/CD5+ CLL cells co-express GLI1 and confocal microscopy showed co-localization of GLI1 and phosphorylated STAT3. Chromatin immunoprecipitation showed that STAT3 protein co-immunoprecipitated GLI1 as well as other STAT3-regulated genes. Transfection of CLL cells with STAT3-shRNA induced a mark decrease in GLI1 levels, suggesting that STAT3 binds to and induces the expression of GLI1 in CLL cells. An electromobility shift assay confirmed that STAT3 binds, and a luciferase assay showed that STAT3 activates the GLI1 gene. Transfection with GLI1-siRNA significantly increased the spontaneous apoptosis rate of CLL cells, suggesting that GLI1 inhibitors might provide therapeutic benefit to patients with CLL.

Myelofibrosis (MF) is a myeloproliferative neoplasm characterized by cytopenia and extramedullary hematopoiesis, resulting in splenomegaly. Multiple pathological mechanisms (e.g., circulating cytokines and genetic alterations, such as JAKV617F mutation) have been implicated in the etiology of MF, but the molecular mechanism causing resistance to JAK2V617F inhibitor therapy remains unknown. Among MF patients who were treated with the JAK inhibitor ruxolitinib, we compared noncoding RNA profiles of ruxolitinib therapy responders versus nonresponders and found miR-543 was significantly upregulated in nonresponders. We validated these findings by reverse transcription–quantitative PCR. in this same cohort, in 2 additional independent MF patient cohorts from the United States and Romania, and in a JAK2V617F mouse model of MF. Both in vitro and in vivo models were used to determine the underlying molecular mechanism of miR-543 in MF. Here, we demonstrate that miR-543 targets the dioxygenases ten-eleven translocation 1 (TET1) and 2 (TET2) in patients and in vitro, causing increased levels of global 5-methylcytosine, while decreasing the acetylation of histone 3, STAT3, and tumor protein p53. Mechanistically, we found that activation of STAT3 by JAKs epigenetically controls miR-543 expression via binding the promoter region of miR-543. Furthermore, miR-543 upregulation promotes the expression of genes related to drug metabolism, including CYP3A4, which is involved in ruxolitinib metabolism. Our findings suggest miR-543 as a potentially novel biomarker for the prognosis of MF patients with a high risk of treatment resistance and as a potentially new target for the development of new treatment options The work in GAC’s laboratory was partly supported by NIH/National Center for Advancing Translational Sciences grant UH3TR00943-01, the NIH/National Cancer Institute (NCI) grant 1 R01 CA182905-01, U54 grant UPR/MDACC Partnership for Excellence in Cancer Research 2016 Pilot Project, Team Department of Defense grant CA160445P1, a Ladies Leukemia League grant, a Chronic Lymphocytic Leukemia Moonshot Flagship project, a Sister Institution Network Fund 2017 grant, and the Estate of C. G. Johnson, Jr. EFM was supported in part by award number P50 CA140388 from the NCI and by the NIH Clinical Research Loan Repayment Program. AMS was supported by Fundação para a Ciência e a Tecnologia through fellowship SFRH/BD/85968/2012. ZJZ’s work was supported by grants from the MPN Foundation and the Oklahoma Center for the Advancement of Science and Technology. DG, MPD, and IBN were supported in part by a Programul Opera¿ional Competitivitate grant nr35/01.09.2016, ID 37_796, titled “Clinical and economical impact of personalized targeted anti-mi-croRNA therapies in reconverting lung cancer chemoresistance” (CANTEMIR). NSDA was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo, BEPE 2016/09349-4.

The cancer-risk-associated rs6983267 single nucleotide polymorphism (SNP) and the accompanying long noncoding RNA CCAT2 in the highly amplified 8q24.21 region have been implicated in cancer predisposition, although causality has not been established. Here, using allele-specific CCAT2 transgenic mice, we demonstrate that CCAT2 overexpression leads to spontaneous myeloid malignancies. We further identified that CCAT2 is overexpressed in bone marrow and peripheral blood of myelodysplastic/myeloproliferative neoplasms (MDS/MPN) patients. CCAT2 induces global deregulation of gene expression by down-regulating EZH2 in vitro and in vivo in an allele-specific manner. We also identified a novel non-APOBEC, non-ADAR, RNA editing at the SNP locus in MDS/MPN patients and CCAT2-transgenic mice. The RNA transcribed from the SNP locus in malignant hematopoietic cells have different allelic composition from the corresponding genomic DNA, a phenomenon rarely observed in normal cells. Our findings provide fundamental insights into the functional role of rs6983267 SNP and CCAT2 in myeloid malignancies.

Background: Prior work from our group has shown that fibrocytes, the cells driving bone marrow (BM) fibrosis in patients with primary myelofibrosis (PMF), are neoplastic (clonal) and derived from monocytes (Verstovsek, J Exp Med 2016). These findings led to the clinical development of PRM-151 (recombinant human pentraxin-2) as an anti-fibrotic agent for patients with myelofibrosis (MF) (Verstovsek, EHA 2019). Our observations were extended by others to show that thrombopoietin receptor (MPL) activation induces fibrocyte differentiation and that blood monocytes highly expressing MPL and signaling lymphocyte activation molecule family member 7 (SLAMF7) were possible fibrocyte precursors (Maekawa, Leukemia 2018). Furthermore, patients with JAK2V617F+ MF have a significantly elevated SLAMF7 high monocyte percentage, which correlates with the JAK2V617F allele burden (Maekawa, Blood 2019). Finally, elotuzumab, a SLAMF7-targeting monoclonal antibody, inhibited the differentiation of MF patient-derived fibrocytes in vitro and romiplostim-induced MF and splenomegaly in vivo. Study design and methods: This is a single-institution, investigator-initiated, pilot phase 2 study of elotuzumab monotherapy in patients with JAK2V617F+ PMF or post-polycythemia vera/essential thrombocythemia MF who need treatment but are not candidates for JAK inhibitor therapy. Baseline BM fibrosis grade must be 2 or 3 per the European consensus (Thiele, Haematologica 2005). Prior JAK inhibitor treatment is permitted. Elotuzumab is dosed intravenously weekly at 10 mg/kg per dose for the first 8 doses, followed by 20 mg/kg every 4 weeks, per the label for its use in multiple myeloma in combination with pomalidomide and dexamethasone. Patients may continue elotuzumab until disease progression or unacceptable toxicity, up to a maximum of 36 cycles. Premedication and management of infusion reactions are carried out according to the elotuzumab package insert. Spleen and liver sizes are measured by palpation and the MPN-SAF-TSS questionnaire (Emanuel, J Clin Oncol 2012) is administered on day 1 of each cycle. Patients receive a BM biopsy at screening and every 6 cycles while on-study. Plasma cytokines are measured at baseline and every 3 cycles while on-study. The primary endpoint is overall response rate according to the revised IWG-MRT-ELN criteria (Tefferi, Blood 2013). A total of 15 patients are planned to be enrolled. Elotuzumab is provided by Bristol-Myers Squibb. Adverse events are graded according to the National Cancer Institute's Common Terminology Criteria for Adverse Events (CTCAE), version 5.0. The method of Thall, Simon and Estey (Thall, Stat Med 1995) is used for toxicity monitoring. Correlative studies: These include quantification of SLAMF7 highCD16 neg circulating monocytes by flow cytometry, measurement of serum interleukin-1 receptor alpha (IL-1Rα) concentrations and correlation of these with each other and with the mutant JAK2 allele burden, culture of human fibrocytes from peripheral blood mononuclear cells (PBMCs) in vitro, engraftment of BM cells from patients in non-obese diabetic, severe combined immunodeficient gamma (NSG) mice, and quantitation of fibrocytes in the BM of participants at baseline and every 6 cycles. Current status: The study (clinicaltrials.gov identifier: NCT04517851) is ongoing; 2 participants have been enrolled and treated thus far. Updated enrollment information will be provided. Disclosures Bose: Astellas: Research Funding; Sierra Oncology: Honoraria; Constellation Pharmaceuticals: Research Funding; Novartis: Honoraria; Celgene Corporation: Honoraria, Research Funding; Incyte Corporation: Honoraria, Research Funding; NS Pharma: Research Funding; Promedior: Research Funding; Blueprint Medicines: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Kartos Therapeutics: Honoraria, Research Funding; CTI BioPharma: Honoraria, Research Funding; Pfizer: Research Funding. Jain: TG Therapeutics: Honoraria; Janssen: Honoraria; Servier: Honoraria, Research Funding; Aprea Therapeutics: Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; Beigene: Honoraria; Adaptive Biotechnologies: Honoraria, Research Funding; Genentech: Honoraria, Research Funding; Precision Biosciences: Honoraria, Research Funding; ADC Therapeutics: Honoraria, Research Funding; Pfizer: Research Funding; Cellectis: Honoraria, Research Funding; Fate Therapeutics: Research Funding; AstraZeneca: Honoraria, Research Funding; Incyte: Research Funding; Pharmacyclics: Research Funding. Pemmaraju: Roche Diagnostics: Consultancy; ASH Communications Committee: Membership on an entity's Board of Directors or advisory committees; ASCO Leukemia Advisory Panel: Membership on an entity's Board of Directors or advisory committees; Samus: Other, Research Funding; Springer Science + Business Media: Other; HemOnc Times/Oncology Times: Membership on an entity's Board of Directors or advisory committees; Dan's House of Hope: Membership on an entity's Board of Directors or advisory committees; DAVA Oncology: Consultancy; Clearview Healthcare Partners: Consultancy; Blueprint Medicines: Consultancy; Protagonist Therapeutics, Inc.: Consultancy; Sager Strong Foundation: Other; Cellectis S.A. ADR: Other, Research Funding; Daiichi Sankyo, Inc.: Other, Research Funding; Plexxicon: Other, Research Funding; CareDx, Inc.: Consultancy; Aptitude Health: Consultancy; MustangBio: Consultancy, Other; Abbvie Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; Celgene Corporation: Consultancy; Stemline Therapeutics, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; LFB Biotechnologies: Consultancy; Novartis Pharmaceuticals: Consultancy, Other: Research Support, Research Funding; Incyte: Consultancy; Affymetrix: Consultancy, Research Funding; Bristol-Myers Squibb Co.: Consultancy; ImmunoGen, Inc: Consultancy; Pacylex Pharmaceuticals: Consultancy. Kantarjian: Amgen: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; NOVA Research: Honoraria; Precision Biosciences: Honoraria; Astra Zeneca: Honoraria; KAHR Medical Ltd: Honoraria; Ipsen Pharmaceuticals: Honoraria; Daiichi-Sankyo: Research Funding; Jazz: Research Funding; Immunogen: Research Funding; BMS: Research Funding; Astellas Health: Honoraria; AbbVie: Honoraria, Research Funding; Ascentage: Research Funding; Novartis: Honoraria, Research Funding; Aptitude Health: Honoraria; Taiho Pharmaceutical Canada: Honoraria. Verstovsek: Blueprint Medicines Corp: Research Funding; Promedior: Research Funding; PharmaEssentia: Research Funding; Protagonist Therapeutics: Research Funding; CTI BioPharma: Research Funding; Celgene: Consultancy, Research Funding; Genentech: Research Funding; NS Pharma: Research Funding; Ital Pharma: Research Funding; Incyte Corporation: Consultancy, Research Funding; Gilead: Research Funding; Sierra Oncology: Consultancy, Research Funding; Roche: Research Funding; AstraZeneca: Research Funding; Novartis: Consultancy, Research Funding; Constellation: Consultancy; Pragmatist: Consultancy. OffLabel Disclosure: Elotuzumab is a monoclonal antibody targeting SLAMF7, previously known as CS-1. It is approved for the treatment of multiple myeloma in combination with an IMiD and dexamethasone. This trial studies it as a single agent in patients with myelofibrosis.

Primary myelofibrosis (MF), post-polycythemia vera (PV) MF, and post-essential thrombocytosis MF are myeloproliferative neoplasms (MPNs) characterized by progressive bone marrow (BM) fibrosis. A recent study suggested that glioma-associated oncogene-1 (GLI1), a downstream effector of the embryonic Hedgehog pathway, is implicated in the pathogenesis of BM fibrosis in MF. Because we and other investigators have found that monocyte-derived fibrocytes, rather than mesenchymal stromal cells (MSCs), induce BM fibrosis in MF, we sought to determine the role of GLI1 in MF fibrocytes. To do this, we analyzed BM biopsy sections of patients with MF using multiplex fluorescence immunohistochemistry and detected high levels of GLI1 in CD45+/CD68+/procollagen I+ fibrocytes and low levels of GLI1 in CD90+/CD105+ MSCs (P=0.009 and P=0.002, respectively). Using immunostaining, RNA in situ hybridization, gene expression, and western immunoblotting analyses of cultured BM fibrocytes or MSCs, we observed significantly higher levels of GLI1 and GLI1-induced matrix metalloproteases (MMP) 2 and 9 in MF fibrocytes than in MF MSCs or normal BM-derived fibrocytes (P

Prognostic scoring systems for primary myelofibrosis (PMF) are not accurate in patients with post-essential thrombocythemia and post-polycythemia vera myelofibrosis (PET-MF; PPV-MF). Given the paucity of data describing the clinical characteristics, disease course and outcomes of these patients, we sought to describe and compare the clinical characteristics and outcomes of 755 patients with PMF, 181 with PPV-MF, and 163 with PET-MF referred to our institution between 1984 and 2013. The median follow-up was 31 months, and 56% (n=616) patients had died. Over an observation period of 3,502 person-years, 11% of patients had progression to AML, with similar rates among groups. The proportion of patients with transfusion dependency (higher in PMF), leukocytosis and systemic symptoms (higher in PPV-MF), and thrombocytopenia (higher in PMF, PPV-MF) differed among groups. Median overall survival (OS) was longest in PET-MF patients (73 mo vs 45 mo (PMF) vs 48 mo (PPV-MF), p

To confirm that neoplastic monocyte-derived collagen- and fibronectin-producing fibrocytes induce bone marrow (BM) fibrosis in primary myelofibrosis (PMF), we injected PMF BM-derived fibrocyte-precursor CD14+/CD34- monocytes into the tail vein of NOD-SCID-γ (NSG) mice. PMF BM-derived CD14+/CD34- monocytes engrafted and induced a PMF-like phenotype with splenomegaly, myeloid hyperplasia with clusters of atypical megakaryocytes, persistence of the JAK2V617F mutation, and BM and spleen fibrosis. As control we used normal human BM-derived CD14+/CD34- monocytes. These monocytes also engrafted and gave rise to normal megakaryocytes that, like PMF CD14+/CD34--derived megakaryocytes, expressed HLA-ABC and human CD42b antigens. Using 2 clonogenic assays we confirmed that PMF and normal BM-derived CD14+/CD34- monocytes give rise to megakaryocyte colony-forming cells, suggesting that a subpopulation BM monocytes harbors megakaryocyte progenitor capacity. Taken together, our data suggest that PMF monocytes induce myelofibrosis-like phenotype in immunodeficient mice and that PMF and normal BM-derived CD14+/CD34- monocytes give rise to megakaryocyte progenitor cells.

We previously demonstrated that peripheral blood (PB) is a reliable source for testing JAK2V617F mutation in patients with myelofibrosis (MF); saliva has also been tested to detect such mutation, however its diagnostic accuracy as compared to PB has not been validated. In this study, we prospectively tested 167 patients with MF for JAK2V617F mutation, using both saliva and PB collected at the same time from each patient. The concordance between the 2 sources was 96%, with a sensitivity of 100% and a specificity of 90%. The only factor associated with false positivity on saliva was ongoing transfusion dependency. JAK2V617F testing using saliva is a simple, non-invasive, and potentially a more reliable method than PB for measuring JAK2 status and assessing V617F allelic burden in patients with transfusion dependency.

Myeloproliferative neoplasms with myelofibrosis (MPN-MF) demonstrate constitutive activation of Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling that responds to treatment with the JAK1 and 2 kinase inhibitor (JAKi) ruxolitinib. However, MPN-MF often progresses (~20%) to secondary acute myeloid leukemia (sAML), where standard induction chemotherapy or ruxolitinib is relatively ineffective, necessitating the development of novel therapeutic approaches. In the present studies, we demonstrate that treatment with BET (bromodomain and extraterminal) protein inhibitor (BETi), for example, JQ1, inhibits growth and induces apoptosis of cultured and primary, patient-derived (PD), post-MPN sAML blast progenitor cells. Reverse-phase protein array, mass-cytometry and Western analyses revealed that BETi treatment attenuated the protein expressions of c-MYC, p-STAT5, Bcl-xL, CDK4/6, PIM1 and IL-7R, whereas it concomitantly induced the levels of HEXIM1, p21 and BIM in the sAML cells. Co-treatment with BETi and ruxolitinib synergistically induced apoptosis of cultured and PD sAML cells, as well as significantly improved survival of immune-depleted mice engrafted with human sAML cells. Although BETi or heat shock protein 90 inhibitor (HSP90i) alone exerted lethal activity, cotreatment with BETi and HSP90i was synergistically lethal against the ruxolitinib-persister or ruxolitinib-resistant sAML cells. Collectively, these findings further support in vivo testing of BETi-based combinations with JAKi and HSP90i against post-MPN sAML cells.

Estrov and collaborators examine the role of fibrocytes in primary myelofibrosis and propose a novel therapeutic approach., Primary myelofibrosis (PMF) is a fatal neoplastic disease characterized by clonal myeloproliferation and progressive bone marrow (BM) fibrosis thought to be induced by mesenchymal stromal cells stimulated by overproduced growth factors. However, tissue fibrosis in other diseases is associated with monocyte-derived fibrocytes. Therefore, we sought to determine whether fibrocytes play a role in the induction of BM fibrosis in PMF. In this study, we show that BM from patients with PMF harbors an abundance of clonal, neoplastic collagen- and fibronectin-producing fibrocytes. Immunodeficient mice transplanted with myelofibrosis patients’ BM cells developed a lethal myelofibrosis-like phenotype. Treatment of the xenograft mice with the fibrocyte inhibitor serum amyloid P (SAP; pentraxin-2) significantly prolonged survival and slowed the development of BM fibrosis. Collectively, our data suggest that neoplastic fibrocytes contribute to the induction of BM fibrosis in PMF, and inhibiting fibrocyte differentiation with SAP may interfere with this process.

Protein arginine methyltransferase 5 (PRMT5) is the major type II PRMT that catalyzes the formation of symmetrical dimethyl arginine (SDMA) on protein substrates and plays important roles in various cellular pathways including tumorigenesis. PRMT5 can methylate histones H3 and H4, thereby leading to repression of tumor suppressor genes at specific loci. PRMT5 also regulates gene expression on a global level by methylating and altering the function of transcription factors such as p53, E2F-1, NF-κB and others. Additionally, PRMT5 has been shown to interact with members of the spliceosome complex and regulate pre-mRNA processing of key target genes. In this study, we describe the in vitro and in vivo activity of PRT543, a novel, potent and selective inhibitor of PRMT5, in hematological and solid tumor models. In a scintillation proximity based radiometric assay, PRT543 inhibited the methyltransferase activity of the PRMT5/MEP50 complex with an IC50 of 10.8 nM. It was also highly selective against a panel of 36 other methyltransferases. PRT543 robustly inhibited cell proliferation in a panel of >50 cancer cell lines, representing both solid tumors and cancers of hematological origin, in a concentration-dependent manner with IC50 values ranging from 10 - 1000 nM. This anti-proliferative activity correlated with a reduction of symmetrically dimethylated SmD3 protein, a known PRMT5 substrate, demonstrating effective inhibition of PRMT5 enzymatic activity in cells. PRT543 demonstrated good oral bioavailability and favorable pharmacokinetic properties. Oral administration of this compound led to significant tumor growth inhibition in several subcutaneous mouse xenograft models including mantle cell lymphoma (Granta-519, Z-138), acute myeloid leukemia (MV4-11, SET2, HEL), small cell lung cancer (NCI-H1048) and bladder cancer (5637) at well-tolerated doses. This in vivo activity was accompanied by a corresponding decrease in symmetrically dimethylated SmD3 in tumor tissue collected from the treated animals. PRT543 was also tested in combination with other approved targeted therapies both in vitro and in vivo. Combining PRT543 with the BCL2 inhibitor, venetoclax, revealed a potent synergistic interaction in models of leukemia and lymphoma. Further ex vivo testing in genetically-defined primary patient tumor samples is ongoing. PRT543 is currently under evaluation in a Phase I clinical trial in patients with advanced solid tumors and hematological malignancies (NCT03886831). Citation Format: Neha Bhagwat, Yang Zhang, Hong Lin, Min Wang, Dave Rominger, Tom Emm, Divya Chugani-Mahtani, Dimitrios Angelis, Rupa Shetty, Raul Leal, William Gowen-MacDonald, Alexander Grego, Juan Luengo, Taghi Manshouri, Friederike Pastore, Ross L. Levine, Srdan Verstovsek, Bruce Ruggeri, Peggy Scherle, Kris Vaddi. Preclinical characterization of PRT543, a potent and selective inhibitor of protein arginine methyltransferase 5 (PRMT5), with broad antitumor activity in in vitro and in vivo models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2915.

Transformation of post-myeloproliferative neoplasms into secondary (s) AML exhibit poor clinical outcome. In addition to increased JAK-STAT and PI3K-AKT signaling, post-MPN sAML blast progenitor cells (BPCs) demonstrate increased nuclear β-catenin levels and TCF7L2 (TCF4) transcriptional activity. Knockdown of β-catenin or treatment with BC2059 that disrupts binding of β-catenin to TBL1X (TBL1) depleted nuclear β-catenin levels. This induced apoptosis of not only JAKi-sensitive but also JAKi-persister/resistant post-MPN sAML BPCs, associated with attenuation of TCF4 transcriptional targets MYC, BCL-2, and Survivin. Co-targeting of β-catenin and JAK1/2 inhibitor ruxolitinib (rux) synergistically induced lethality in post-MPN sAML BPCs and improved survival of mice engrafted with human sAML BPCs. Notably, co-treatment with BET protein degrader ARV-771 and BC2059 also synergistically induced apoptosis and improved survival of mice engrafted with JAKi-sensitive or JAKi-persister/resistant post-MPN sAML cells. These preclinical findings highlight potentially promising anti-post-MPN sAML activity of the combination of β-catenin and BETP antagonists against post-MPN sAML BPCs.

Although most patients with myelofibrosis (MF) derive benefit from ruxolitinib, some are refractory, have a suboptimal response, or quickly lose their response. To identify genes that may predict response to ruxolitinib, we performed targeted next-generation sequencing (NGS) of a panel of 28 genes recurrently mutated in hematologic malignancies in a cohort of patients with MF who were treated with ruxolitinib in a phase 1/2 study. We also tested for CALR deletions by standard polymerase chain reaction methods. Ninety-eight percent of patients had a mutation in ≥1 gene. Seventy-nine (82.1%) patients had the JAK2(V617F) mutation, 9 (9.5%) had CALR mutations (7 type 1, 2 type 2), 3 (3.1%) had MPL mutations, and 4 (4.2%) were negative for all 3. ASXL1/JAK2 and TET2/JAK2 were the most frequently comutated genes. Mutations in NRAS, KRAS, PTPN11, GATA2, TP53, and RUNX1 were found in

Therapy-related myeloid neoplasms, such as myelodysplastic syndromes (t-MDSs) and acute myeloid leukemia (t-AML), account for 10% to 20% of all myeloid neoplasms and are recognized as a distinct subgroup according to 2008 World Health Organization (WHO) criteria.[1][1] Although t-MDS/t-AML are often

Despite significant improvements in the signs and symptoms of myelofibrosis (MF), and possible prolongation of patients' survival, some have disease that is refractory to ruxolitinib and many lose their response over time. Furthermore, patients with ≥3 mutations are less likely to respond to ruxolitinib. Here we describe outcomes after ruxolitinib discontinuation in MF patients enrolled in a phase 1/2 study at our center. After a median follow-up of 79 months, 86 patients had discontinued ruxolitinib (30 of whom died while on therapy). The median follow-up after ruxolitinib discontinuation for the remaining 56 patients was 32 months, with median survival after discontinuation of 14 months. Platelets

In most patients with primary myelofibrosis, one of three mutually exclusive somatic mutations is detected. In approximately 60% of patients, the Janus kinase 2 gene is mutated, in 20%, the calreticulin gene is mutated, and in 5%, the myeloproliferative leukemia virus gene is mutated. Although patients with mutated calreticulin or myeloproliferative leukemia genes have a favorable outcome, and those with none of these mutations have an unfavorable outcome, prognostication based on mutation status is challenging due to the heterogeneous survival of patients with mutated Janus kinase 2. To develop a prognostic model based on mutation status, we screened primary myelofibrosis patients seen at the MD Anderson Cancer Center, Houston, USA, between 2000 and 2013 for the presence of Janus kinase 2, calreticulin, and myeloproliferative leukemia mutations. Of 344 primary myelofibrosis patients, Janus kinase 2V617F was detected in 226 (66%), calreticulin mutation in 43 (12%), and myeloproliferative leukemia mutation in 16 (5%) ; 59 patients (17%) were triple-negatives. A 50% cut-off dichotomized Janus kinase 2-mutated patients into those with high Janus kinase 2V617F allele burden and favorable survival and those with low Janus kinase 2V617F allele burden and unfavorable survival. Patients with a favorable mutation status (high Janus kinase 2V617F allele burden/myeloproliferative leukemia/calreticulin mutation) and aged 65 years or under had a median survival of 126 months. Patients with one risk factor (low Janus kinase 2V617F allele burden/triple-negative or age >65 years) had an intermediate survival duration, and patients aged over 65 years with an adverse mutation status (low Janus kinase 2V617F allele burden or triple- negative) had a median survival of only 35 months. Our simple and easily applied age- and mutation status-based scoring system accurately predicted the survival of patients with primary myelofibrosis.

The transcription factor glioma associated oncogene (Gli)-1 is a downstream effector of the Hedgehog (HH) signaling pathway. The HH pathway plays a critical role in embryonic development, and in maintenance of stem cells, tissue homeostasis, hematopoiesis, and immune response in the adult organism. Constitutive activation of the HH pathway has been implicated in the development of various cancer types. A recent study of blood samples from 841 treatment-naïve CLL patients identified activated Gli1 in 49%, and mutations in HH pathway genes, that induce activation of Gli1, in 11% of the patients (Ghia et al. Blood 1019). However what induces the expression of Gli1 in CLL cells that do not carry a mutation(s) in the HH pathway has not been elucidated. Because inhibition of the Janus kinase (JAK)-signal transducer and activator of transcription (STAT)-3 pathway in liver cells inhibits the transcription of Gli1 (Zhang et al. J Cell Mol Med 2018), and because in CLL cells STAT3 is constitutively phosphorylated on serine 727 residues and activates genes known to be activated by phosphotyrosine STAT3, we postulated that constitutively activated STAT3 induces the expression of Gli1 in CLL cells. To test our hypothesis we first performed western immunoblotting and found high levels of Gli1 in cell lysates from 6 of 7 CLL patients but not in normal B cells, and using flow cytometry we found that 76% of CD5+/CD19+ CLL cells expressed intracellular Gli1 and that intracellular phosphoserine STAT3 and Gli1 are co-expressed in 70.4% of CLL cells. Then, using chromatin immunoprecipitation (ChIP) we found that STAT3 protein co-immunoprecipitated with DNA of Gli1 and of the STAT3-target genes C-Myc, ROR1, VEGF, and STAT3. Using sequence analysis we identified in the Gli1 gene promoter 5 γ-interferon activation sequence (GAS)-like elements, known as putative STAT3-binding sites, spanning within 930 base pairs upstream the Gli1 start codon. To determine which putative binding site binds STAT3 we designed 5 different probes. Using ChIP we found that STAT3 protein co-immunoprecipitated 4 of 5 STAT3-putative binding sites at high affinity. Then, using an electromobility shift assay (EMSA) of CLL cell nuclear extracts from 3 different patients we detected STAT3-Gli1 DNA complexes with DNA probes designed to detect the PTX3 gene promoter STAT3-binding sites and found that anti-STAT3 antibodies significantly attenuated the binding, confirming that STAT3 binds to the Gli1 gene promoter. To further confirm these findings, we transfected CLL cells with STAT3-short hairpin (sh)RNA and found that STAT3-shRNA significantly downregulated STAT3 and Gli1 mRNA levels. Because Gli1 expression was associated with disease progression (Ghia et al. Blood 1019), we wondered whether Gli1 provides CLL cells with survival advantage and whether inhibition of Gli1 would affect the viability of CLL cells. To answer this question we transfected CLL cells from two different treatment-naïve CLL patients with Gli1 small interfering (si)RNA and, using annexin V/PI, found that Gli1-siRNA induced apoptosis of CLL cells. To further confirm these data we incubated CLL cells with GANT61, a small molecule that inhibits Gli1, and, as previously reported, found that GANT61 increases the spontaneous apoptosis rate of CLL cells, suggesting that inhibition of Gli1 might benefit patients with CLL. In conclusion, we found that constitutively phosphorylated STAT3 activates the Gli1 gene promoter and induced the production of Gli1 protein in CLL cells. In addition, we found that downregulation of Gli1 expression or inhibition of Gli1 induced apoptosis of CLL cells, suggesting that Gli1 is a target for therapy of CLL. Disclosures Burger: BeiGene: Research Funding; Janssen Pharmaceuticals: Consultancy, Honoraria; AstraZeneca: Honoraria; Aptose Biosciences, Inc: Research Funding; Pharmacyclics, an AbbVie company: Research Funding; Gilead Sciences: Research Funding. Bose:Blueprint Medicine Corporation: Consultancy, Research Funding; Incyte Corporation: Consultancy, Research Funding, Speakers Bureau; Celgene Corporation: Consultancy, Research Funding; Kartos: Consultancy, Research Funding; Constellation: Research Funding; Pfizer: Research Funding; Astellas: Research Funding; NS Pharma: Research Funding; Promedior: Research Funding; CTI BioPharma: Research Funding. Thompson:Gilead: Consultancy, Honoraria; Genentech: Consultancy, Honoraria; Pharmacyclics: Research Funding; Pfizer: Research Funding; Amgen: Consultancy, Research Funding; AbbVie: Research Funding. Jain:Pharmacyclics, an AbbVie company: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Verastem: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Precision Biosciences: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive Biotechnologies: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cellectis: Research Funding; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; ADC Therapeutics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Research Funding; AstraZeneca: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Servier: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Verstovsek:NS Pharma: Research Funding; Celgene: Consultancy, Research Funding; Incyte: Research Funding; Roche: Research Funding; Genetech: Research Funding; Blueprint Medicines Corp: Research Funding; Novartis: Consultancy, Research Funding; Sierra Oncology: Research Funding; Pharma Essentia: Research Funding; Astrazeneca: Research Funding; Ital Pharma: Research Funding; Protaganist Therapeutics: Research Funding; Constellation: Consultancy; Pragmatist: Consultancy; CTI BioPharma Corp: Research Funding; Gilead: Research Funding; Promedior: Research Funding. Wierda:Gilead Sciences: Research Funding; Cyclcel: Research Funding; Sunesis: Research Funding; Acerta Pharma Inc: Research Funding; AbbVie: Research Funding; KITE pharma: Research Funding; Xencor: Research Funding; Janssen: Research Funding; GSK/Novartis: Research Funding; Miragen: Research Funding; Genentech: Research Funding; Juno Therapeutics: Research Funding; Pharmacyclics LLC: Research Funding; Loxo Oncology Inc.: Research Funding; Oncternal Therapeutics Inc.: Research Funding.

Myelofibrosis (MF) is characterized by constitutive activation of the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway and progressive bone marrow (BM) fibrosis. We previously found that the BM fibrosis in MF is induced by clonal monocyte-derived fibrocytes. However, whether or how the JAK-STAT pathway affects MF fibrocyte differentiation and maturation has not been elucidated. A recent study suggested that the transcription factor glioma-associated oncogene homolog 1 (Gli1) plays a significant role in the induction of BM fibrosis in MF (Schneider et al. Cell Stem Cell 2017). We have recently found that MF BM fibrocytes express high levels of Gli1 both in situ and in vitro (ASH 2018: Abstract 1793). Therefore, we sought to determine whether Gli1 affects the differentiation and maturation of MF fibrocytes and whether Gli1 expression is induced by the constitutively activated JAK-STAT pathway. To test our hypothesis, we first cultured normal donor and MF patients' BM CD14+ monocytes in fibrocyte differentiation-supporting medium and then transfected them using Gli1-siRNA. After 14 days we quantitated the number of mature fibrocytes using F-actin and DAPI staining. We found that the numbers of BM monocyte-derived fibrocytes in cultures containing Gli1-siRNA were significantly lower compared to mock-transfected cells. To study downstream effects of Gli1 silencing, we transfected mature fibrocytes with Gli1-siRNA and found that the expression of Gli1 was reduced by 137-fold (P Because JAK-STAT pathway is constitutively activated in MF patients' fibrocytes, we sought to determine whether Gli1 expression is induced by STAT3. Using sequence analysis we identified 5 putative STAT3-binding sites in the Gli1 gene promoter region and designed probes that target these sites. Using chromatin immunoprecipitation (ChIP) assay we found that STAT3 protein co-precipitated all 5 STAT3-putative binding sites at high affinity. Then, by performing an electromobility shift assay (EMSA) of the fibrocyte nuclear extracts using DNA probes against Gli1 gene promoter STAT3-binding sites we detected STAT3-Gli1 DNA complexes, suggesting that STAT3 binds to the Gli1 gene promoter. We further confirmed these findings using anti-STAT3 antibodies which significantly attenuated the binding. Finally, we transfected MF fibrocytes with STAT3-siRNA and found that STAT3 siRNA significantly decreased Gli1 mRNA levels. In conclusion, we found that STAT3, phosphorylated by constitutively activated JAK2, induces the expression of Gli1 which in turn promotes the differentiation and maturation of MF fibrocytes. Downregulation of Gli1 levels halted fibrocyte development, suggesting that inhibition of Gli1 might alleviate BM fibrosis in patients with MF. Disclosures Verstovsek: Incyte: Research Funding; Roche: Research Funding; NS Pharma: Research Funding; Celgene: Consultancy, Research Funding; Gilead: Research Funding; Promedior: Research Funding; CTI BioPharma Corp: Research Funding; Genetech: Research Funding; Blueprint Medicines Corp: Research Funding; Novartis: Consultancy, Research Funding; Sierra Oncology: Research Funding; Pharma Essentia: Research Funding; Astrazeneca: Research Funding; Ital Pharma: Research Funding; Protaganist Therapeutics: Research Funding; Constellation: Consultancy; Pragmatist: Consultancy.

Human pentraxins are a family of proteins with a unique pentameric structure. Unlike C-reactive protein (CRP), serum amyloid P (SAP) and pentraxin-3 (PTX3) play an opposite role in tissue remodeling. PTX3 induces whereas SAP inhibits the differentiation of CD14+ monocytes into fiborcytes. While in patients with CLL CRP levels are high and were found to be associated with poor overall survival (OS) (Herishanu et al. Ann Med 2017), little is known about the plasma levels or clinical significance of other pentraxins in CLL. Therefore, we obtained plasma sample from 36 randomly chosen treatment-naïve CLL patients and 12 age-matched healthy individuals and, using an enzyme linked immuno-sorbent assay, found that PTX3, CRP and SAP plasma levels were significantly higher in CLL patients than in healthy individuals (P Whereas CRP and SAP are produced by the liver, PTX3 was found to be released from macrophages and endothelial cells. Because in patients with CLL is characterized by an increased number of circulating CLL cells, we wondered whether CLL cells produce PTX3. Using flow cytometry we found that approximately 50% of CD5+/CD19+ CLL cells co-expressed intracellular PTX3, and using Western immunoblotting we detected PTX3 protein in 7 out of 7 CLL patients' cell extracts but not in normal B cells. Therefore we sought to determine why CLL cells produce PTX3. In MF, characterized by constitutive activation of the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway, high PTX3 levels constitute an independent indicator of disease burden, clonal expansion and OS (Veletic, et al. Br J Haemat 2019). In CLL STAT3 is constitutively phosphorylated on serine 727 residues and activates genes known to be activated by phosphotyrosine STAT3. Therefore we postulated that phosphoserine STAT3 induces the production of PTX3 in CLL cells. To test our hypothesis we performed western immunoblotting and found PTX3 protein in 6 of 6 CLL patients' cell extracts, and by using flow cytometry we found that intracellular phosphoserine STAT3 and PTX3 are co-expressed in 77% of CLL cells. Then, using chromatin immunoprecipitation (ChIP) we found that STAT3 protein co-immunoprecipitated with DNA of PTX3 and with DNA of the STAT3-target genes C-Myc, ROR1, VEGF, and STAT3. Sequence analysis of the PTX3 gene promoter identified four γ-interferon activation sequence (GAS)-like elements, known as putative STAT3-binding sites spanning within 630 base pairs upstream the PTX3 start codon. To determine which putative binding site binds STAT3 we designed three different probes. Using ChIP we found that STAT3 protein co-immunoprecipitated all 3 STAT3-putative binding sites at high affinity. Similar results, obtained with IL-6-stimulated MM1 cells, further confirmed these findings. Then, using an electromobility shift assay (EMSA) of CLL cell nuclear extracts from 3 different patients we detected STAT3-PTX3 DNA complexes with DNA probes, designed to detect the PTX3 gene promoter binding sites, and found that anti-STAT3 antibodies significantly attenuated the binding, confirming that STAT3 binds to the PTX3 gene promoter. To further confirm these data, we transfected CLL cells with STAT3-short hairpin (sh)RNA and found that STAT3-shRNA significantly downregulated STAT3 and PTX3 mRNA levels. In conclusion, we found that constitutively activated STAT3 activates the PTX3 gene and induces the production of high levels of PTX3 in patients with CLL. PTX3, known to induce differentiation of CD14+ monocytes into fibrocytes, enhances the production of BM fibrocytes that induce BM reticulin fibrosis in patients with CLL. Disclosures Burger: Aptose Biosciences, Inc: Research Funding; Gilead Sciences: Research Funding; Pharmacyclics, an AbbVie company: Research Funding; Janssen Pharmaceuticals: Consultancy, Honoraria; BeiGene: Research Funding; AstraZeneca: Honoraria. Bose:Incyte Corporation: Consultancy, Research Funding, Speakers Bureau; Celgene Corporation: Consultancy, Research Funding; Blueprint Medicine Corporation: Consultancy, Research Funding; Kartos: Consultancy, Research Funding; Constellation: Research Funding; Pfizer: Research Funding; Astellas: Research Funding; NS Pharma: Research Funding; Promedior: Research Funding; CTI BioPharma: Research Funding. Thompson:Gilead: Consultancy, Honoraria; Genentech: Consultancy, Honoraria; Pharmacyclics: Research Funding; Pfizer: Research Funding; Amgen: Consultancy, Research Funding; AbbVie: Research Funding. Jain:AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Servier: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cellectis: Research Funding; Verastem: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Precision Biosciences: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive Biotechnologies: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Research Funding; ADC Therapeutics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics, an AbbVie company: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Verstovsek:Protaganist Therapeutics: Research Funding; Constellation: Consultancy; Pragmatist: Consultancy; Incyte: Research Funding; Roche: Research Funding; NS Pharma: Research Funding; Celgene: Consultancy, Research Funding; Gilead: Research Funding; Promedior: Research Funding; CTI BioPharma Corp: Research Funding; Genetech: Research Funding; Blueprint Medicines Corp: Research Funding; Novartis: Consultancy, Research Funding; Sierra Oncology: Research Funding; Pharma Essentia: Research Funding; Astrazeneca: Research Funding; Ital Pharma: Research Funding. Wierda:Cyclcel: Research Funding; AbbVie: Research Funding; Juno Therapeutics: Research Funding; KITE pharma: Research Funding; Genentech: Research Funding; Pharmacyclics LLC: Research Funding; Acerta Pharma Inc: Research Funding; Loxo Oncology Inc.: Research Funding; Sunesis: Research Funding; Oncternal Therapeutics Inc.: Research Funding; Xencor: Research Funding; Janssen: Research Funding; Gilead Sciences: Research Funding; GSK/Novartis: Research Funding; Miragen: Research Funding.

Hemopoietic progenitor cells (HPC) from myeloproliferative neoplasms (MPN) such as myelofibrosis commonly express mutant JAK2-V617F or other mutations that are associated with increased activities of JAK-STAT5/3, RAS/RAF/MAPK, and PI3K/AKT/mTOR pathways. This confers proliferative and survival advantage on the MPN HPCs. Treatment with JAK tyrosine kinase inhibitor (TKI), for example, TG101209, TG101348 (SAR302503), or INCB018424 (ruxolitinib), inhibits mutant JAK2-mediated signaling. Although effective in reducing constitutional symptoms and splenomegaly, treatment with JAK-TKI does not ameliorate myelofibrosis or significantly improve survival of patients with advanced myelofibrosis. Here, we show that treatment with the dual phosphoinositide-3-kinase (PI3K)/AKT and mTOR inhibitor BEZ235 attenuated PI3K/AKT and mTOR signaling, as well as induced cell-cycle growth arrest and apoptosis of the cultured human JAK2-V617F-expressing HEL92.1.7 (HEL), UKE1 cells, and primary CD34+ myelofibrosis (MF)-MPN cells. Treatment with BEZ235 also induced significant apoptosis of the JAK2-TKI resistant HEL/TGR cells that were selected for resistance against JAK-TKI. Cotreatment with BEZ235 and JAK2-TKI (TG101209 and SAR302503) synergistically induced lethal activity against the cultured and primary CD34+ MPN cells while relatively sparing the normal CD34+ HPCs. These findings create a compelling rationale to determine the in vivo activity of dual PI3K/mTOR inhibitors in combination with JAK inhibitors against myelofibrosis HPCs. Mol Cancer Ther; 12(5); 577–88. ©2013 AACR.

Chronic exposure to JAK2 inhibitors leads to reactivation of downstream signalling through the formation of heterodimers between JAK2 and other JAK kinases in myeloproliferative neoplasms, which can be overcome with Hsp90 inhibitors. Mutations in JAK kinases, in particular JAK2, are frequent in some malignancies and JAK inhibitors have been trialled for example in patients with myeloproliferative neoplasms (MPNs). Here, Ross Levine and colleagues demonstrate that MPN cells can persist under conditions of chronic JAK2 inhibition, because JAK2 forms a heterodimer with other JAK kinases, leading to persistent JAK2 activation. This mode of drug 'persistence' seems to occur in patients treated with JAK2 inhibitor. Therapeutic approaches that induce JAK2 degradation may therefore be more effective than treatment with JAK2 inhibitors alone. The identification of somatic activating mutations in JAK2 (refs 1–4) and in the thrombopoietin receptor gene (MPL)5 in most patients with myeloproliferative neoplasm (MPN) led to the clinical development of JAK2 kinase inhibitors6,7. JAK2 inhibitor therapy improves MPN-associated splenomegaly and systemic symptoms but does not significantly decrease or eliminate the MPN clone in most patients with MPN. We therefore sought to characterize mechanisms by which MPN cells persist despite chronic inhibition of JAK2. Here we show that JAK2 inhibitor persistence is associated with reactivation of JAK–STAT signalling and with heterodimerization between activated JAK2 and JAK1 or TYK2, consistent with activation of JAK2 in trans by other JAK kinases. Further, this phenomenon is reversible: JAK2 inhibitor withdrawal is associated with resensitization to JAK2 kinase inhibitors and with reversible changes in JAK2 expression. We saw increased JAK2 heterodimerization and sustained JAK2 activation in cell lines, in murine models and in patients treated with JAK2 inhibitors. RNA interference and pharmacological studies show that JAK2-inhibitor-persistent cells remain dependent on JAK2 protein expression. Consequently, therapies that result in JAK2 degradation retain efficacy in persistent cells and may provide additional benefit to patients with JAK2-dependent malignancies treated with JAK2 inhibitors.

Leukemic transformation (LT) of myeloproliferative neoplasms (MPNs) is associated with a poor prognosis and resistance to therapy. Although previous candidate genetic studies have identified mutations in MPN patients who develop acute leukemia, the complement of genetic abnormalities in MPN patients who undergo LT is not known nor have specific molecular abnormalities been shown to have clinical relevance in this setting. We performed high-throughput resequencing of 22 genes in 53 patients with LT after MPN to characterize the frequency of known myeloid mutations in this entity. In addition to JAK2 and TET2 mutations, which occur commonly in LT after MPN, we identified recurrent mutations in the serine/arginine-rich splicing factor 2 (SRSF2) gene (18.9%) in acute myeloid leukemia (AML) transformed from MPNs. SRSF2 mutations are more common in AML derived from MPNs compared with LT after myelodysplasia (4.8%) or de novo AML (5.6%), respectively (P = .05). Importantly, SRSF2 mutations are associated with worsened overall survival in MPN patients who undergo LT in univariate (P = .03; HR, 2.77; 95% CI, 1.10-7.00) and multivariate analysis (P < .05; HR, 2.11; 95% CI, 1.01-4.42). These data suggest that SRSF2 mutations contribute to the pathogenesis of LT and may guide novel therapeutic approaches for MPN patients who undergo LT.

Background: Primary myelofibrosis (PMF), post-polycythemia vera MF (post-PV MF) and post-essential thrombocythemia MF (post-ET MF) are characterized by expansion of the neoplastic clone and by progressive bone marrow (BM) fibrosis. Like in other hematologic malignancies, in most patients with MF the immune system is significantly deregulated: MF patients' plasma cytokine and chemokine levels are markedly increased and their normal T cell subset distribution is significantly altered. Although treatment with the Janus kinase (JAK)-1/2 inhibitor ruxolitinib significantly decreases cytokine/chemokine levels, reduces spleen size, and improves symptoms and quality of life, it does not reverse BM fibrosis nor does it halt the propagation of the neoplastic clone. The T cell immune checkpoint programmed cell death protein-1 (PD-1) promotes immune tolerance by binding to the tumor's cell surface PD-1 ligand (PD-L1). Whereas the importance of T cell-mediated immune tolerance in MF has been documented and trials evaluating clinical benefits of PD1/PD-L1 checkpoint inhibition are ongoing, little is known about the effect of ruxolitinib on PD-1 expression in T cell subsets. Therefore we systematically analyzed MF patients circulating T cells' surface marker expression prior to and during ruxolitinib treatment. Methods: Peripheral blood cells were obtained from well-characterized PMF, post-PV MF and post-ET MF patients prior to and during the course of treatment with ruxolitinib (n=47) and, as control, from age-matched healthy donors (n=28). The proportion of PD-1-expressing CD4+ and CD8+ cells was assessed using multiparameter flow cytometry. Naïve, central memory, effector memory, and effector T cell subsets were defined based on CD45RO and CD27 cell surface antigen expression. Results: A significantly high number of circulating T cells co-expressing CD4+/PD-1+ and CD8+/PD-1+ was found in MF patients compared to age-matched healthy individuals (5.3±4.1% vs. 3.4±1.7%, P=0.028; 7.1±4.4% vs. 3.8±2.3%, P=0.001). Whereas MF patients' naïve T cells harbored an increased number of cells co-expressing CD8+/PD-1+ (P=0.007), but not CD4+/PD-1+, their T central memory cells had a high proportion of cells co-expressing CD4+/PD-1+ and CD8+/PD-1+ (P Conclusions: In patients with MF, circulating T cells express high levels of PD-1. While not restricted to a particular stage of T cell differentiation, the correlation between PD-1-expressing T cells and distinct clinical parameters suggests that increased PD-1 levels might induce immune exhaustion in T cell subsets in different ways. Although ruxolitinib significantly inhibits the JAK1/2 signaling pathway in a variety of hematopoietic cells, thereby lowering cytokine/chemokine levels in almost all MF patients, treatment with ruxolitinib did not affect PD-1 expression nor did it alter its distribution among the T cell subsets. Yet, the proportion of PD-1-expressing CD4+ and CD8+ cells was markedly reduced in patients who experienced a superior response to ruxolitinib as assessed by significant spleen size reduction. How disease burden and MF microenvironment affect PD-1 expression in T cells of patients with MF warrants further investigation. Disclosures Verstovsek: Incyte: Consultancy; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees; Italfarmaco: Membership on an entity's Board of Directors or advisory committees.

Background: Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by myeloid cell proliferation and progressive bone marrow (BM) fibrosis. The degree of the BM fibrosis correlates with unfavorable clinical characteristics and short survival. Whereas it was thought that BM fibrosis in PMF is secondary, caused by mesenchymal stromal cells (MSC) stimulated by cytokine-producing neoplastic megakaryocytes and platelets, we have recently found that BM fibrosis in PMF is induced by neoplastic monocyte-derived fibrocytes which, like MSC, produce collagen-I, collagen-III and fibronectin. Using a PMF immunodeficient mouse model, we demonstrated that inhibition of monocyte differentiation into fibrocytes ameliorates BM fibrosis. Although an increased number of fibrocytes was detected in the BM of patients with PMF, the mechanisms that induce collagen overproduction are still unknown. Glioma oncogene homolog 1 (Gli1), a transcription factor of the hedgehog signaling pathway, activates genes affecting cell migration and tissue fibrosis. Gene expression analyses identified matrix metalloprotease 9 (MMP9) as a key effector of Gli1 that promotes fibrocyte migration into sites of tissue injury. A recent study demonstrated that knockout or inhibition of Gli1 alleviates BM fibrosis. Since it was thought that BM fibrosis is induced by MSC, the function of other BM-derived Gli1-expressingcells was not investigated. Because fibrocytes induce BM fibrosis in PMF, we sought to evaluate the levels of Gli1 in PMF BM cells and in BM-derived fibrocytes and MSC. Methods: BM trephine biopsies and aspirates were obtained from randomly selected PMF patients and healthy controls. Opal multiplex fluorescence IHC was performed on tissue sections using fibrocyte (CD45, CD68, procollagen type I)- and MSC (CD90, CD105)-specific monoclonal antibodies in combination with validated anti-Gli1 antibodies. Single BM cells were classified based on image pattern analysis and their Gli1 signal intensity was assessed. In addition, Low-density cells from BM aspirates were cultured using fibrocyte and MSC culture assays. Following confirmatory immunostaining analysis, RNA was extracted and analyzed using quantitative RT-PCR. Results: Gli1 intensity in fibrocytes of PMF BM biopsies was significantly higher than Gli1 intensity in fibrocytes of healthy control BM biopsies (median: 11.11 vs. 7.16 counts per cell, P=0.02). The median Gli1 signal intensity/cell was 4.3 counts higher in PMF fibrocytes than in PMF MSC (95%CI: 0.5-11.75; P=0.033), whereas Gli1 intensity/cell of PMF MSC was similar to that of normal BM MSC. Similarly, Gli1 mRNA levels were significantly higher in cultured PMF BM-derived fibrocytes than in cultured normal BM-derived fibrocytes (7.16±1.35 vs. 1±0.55, P=0.007). In addition, mRNA levels were higher in PMF BM-derived fibrocytes than in PMF BM-derived MSC (7.16±1.35 vs. 1.36±0.23, P=0.006). Downstream activity of Gli1 in cultured cells was assessed by quantitating the expression of the MMP9 gene. Remarkably, PMF BM-derived fibrocytes expressed significantly higher MMP9 mRNA levels compared to normal BM-derived fibrocytes (12.47±2.25 vs. 1±0.45, P=0.003). In contrast, in PMF BM-derived MSC MMP9 mRNA levels were only 0.03±0.01, 12-fold lower than those of PMF BM-derived fibrocytes (P Conclusions: Because Gli1 protein is invariably overrepresented in fibrocytes but not in MSC and Gli1, while MMP9 mRNA levels are higher in PMF BM-derived fibrocytes than in MSC, and considering previous Gli1 gene knockout studies, it is reasonable to assume that Gli1+ BM fibrocytes rather than MSC are the major contributors to the induction of BM fibrosis in PMF. Disclosures Verstovsek: Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Incyte: Consultancy; Italfarmaco: Membership on an entity's Board of Directors or advisory committees.

Introduction: PRM-151, a recombinant human pentraxin-2 molecule, has been shown to prevent and reverse fibrosis in animal models of myelofibrosis (MF) by postulated targeting differentiation of fibrocytes (essential cells in fibrotic process) from monocytes. In the first stage of a two-stage trial, 27 patients (pts) with primary myelofibrosis (PMF), post-essential thrombocythemia/polycythemia vera (post-ET/PV) MF, and Grade 2 or 3 bone marrow fibrosis (BMF) received PRM-151 10 mg/kg IV ± ruxolitinib (RUX) for 6 cycles (24 weeks). A reduction in BMF, decrease in symptoms (MPN-SAF Total Symptom Score [TSS]), and a reduction of palpable splenomegaly were observed, along with a favorable safety profile. Pts experiencing clinical benefit were allowed to continue beyond 24 weeks in an open label extension (OLE) study. Here, we report interim efficacy and safety data for 18 pts enrolled in the OLE, who had been treated for up to 35 cycles (140 weeks). Methods: All pts in the OLE received a monthly infusion of PRM-151 10 mg/kg IV. Pts receiving PRM-151 alone or in combination with RUX in the main study continued with their respective drugs. Safety and hematology parameters were assessed monthly, and MPN-SAF TSS Q3 months. BM biopsies were obtained at baseline, end of main study, and at physician discretion in the OLE. These were evaluated centrally by two independent, blinded hematopathologists for the degree of reticulin and collagen fibrosis. Immunostaining for fibrocytes was performed on available biopsies. The primary objective of the OLE was to assess safety and efficacy of long-term administration of PRM-151. Results: Of the 18 pts enrolled in the OLE, 9 received PRM-151 as single agent, and 9 in conjunction with RUX. Baseline characteristics included median age of 66 years (51-78); 8 pts were DIPSS Int-1, 9 Int-2, and 1 high-risk; 6 (33%) had Hgb < 100 g/L, 12 (67%) had PLT < 100 x 109/L; 7 (39%) had PLT Conclusions: This long-term follow-up study of 18 pts with MF who received PRM-151 for a median of 31 months showed the drug to be well tolerated. An overall improvement in BM reticulin and collagen fibrosis grade, as well as reductions in MPN-SAF TSS and splenomegaly were observed. In a subset of pts for whom bone marrow fibrocyte immunostaining data were available, a mean reduction in fibrocyte counts was observed, as well as improved bone marrow reticulin and collagen fibrosis grade. These data warrant confirmation in a larger controlled study. Figure. Figure. Disclosures Verstovsek: Incyte: Consultancy; Italfarmaco: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees. Pozdnyakova:Promedior, Inc.: Consultancy. Mesa:Promedior: Research Funding; Incyte: Research Funding; Ariad: Consultancy; Novartis: Consultancy; Celgene: Research Funding; Galena: Consultancy; Gilead: Research Funding; CTI: Research Funding. Foltz:Incyte: Research Funding; Promedior: Research Funding; Gilead: Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Mascarenhas:Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Merck: Research Funding; Roche: Research Funding; CTI Biopharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; Promedior: Research Funding. Ritchie:Astellas Pharma: Research Funding; NS Pharma: Research Funding; Pfizer: Consultancy, Research Funding; Bristol-Myers Squibb: Research Funding; ARIAD Pharmaceuticals: Speakers Bureau; Novartis: Consultancy, Other: Travel, Accommodations, Expenses, Research Funding, Speakers Bureau; Celgene: Consultancy, Other: Travel, Accommodations, Expenses, Speakers Bureau; Incyte: Consultancy, Speakers Bureau. Palmer:Novartis: Research Funding. Kremyanskaya:Incyte: Research Funding. van den Blink:Promedior, Inc.: Employment. Gupta:Promedior, Inc.: Employment. Gotlib:Deciphera: Consultancy, Honoraria, Research Funding; Incyte: Consultancy, Honoraria, Research Funding; Kartos: Consultancy; Promedior: Research Funding; Gilead: Consultancy, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Blueprint Medicines: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.

CD52 and CD20 antigens are important therapeutic targets for the monoclonal antibodies (mAbs) alemtuzumab and rituximab respectively. Circulating CD52 (cCD52) and CD20 (cCD20) have prognostic utility in lymphoid malignancies. The efficacy of mAb therapy in patients with chronic lymphocytic leukaemia (CLL) may be adversely affected by cCD52 or cCD20. In this report, blood and bone marrow (BM) cCD52 and cCD20 were measured at response assessment in previously treated (N = 235) patients with CLL who received fludarabine, cyclophosphamide, and rituximab (FCR). Univariate and multivariate statistical models evaluated correlations of pre- and response variables with progression-free (PFS) and overall survival (OS). Response variables included 1996 National Cancer Institute-Working Group (NCI-WG) response, polymerase chain reaction (PCR) for immunoglobulin heavy chain (IGHV) in BM, and cCD52 and cCD20 levels (blood and BM) at response assessment. Using multivariate analysis, response blood and BM cCD52, blood cCD20, and NCI-WG response were significant independent predictors of PFS. At the time of response assessment, BM cCD52 correlated with OS in univariate analysis. cCD52 and cCD20, therefore appear useful in predicting survival and may be important for monitoring patients following salvage FCR (fludarabine, cyclophosphamide, rituximab) therapy. These data further indicate that plasma may be a good target to evaluate for minimal residual disease using cCD52/cCD20 levels.

The oncogenetic events that transform chronic myeloproliferative neoplasms (MPN) to acute myeloid leukemias (AML) are not well characterized. We investigated the role of several genes implicated in leukemic transformation by mutational analysis of 63 patients with AML secondary to a preexisting MPN (sAML). Frequent mutations were identified in TET2 (26.3%), ASXL1 (19.3%), IDH1 (9.5%), and JAK2 (36.8%) mutations in sAML, and all possible mutational combinations of these genes were also observed. Analysis of 14 patients for which paired samples from MPN and sAML were available showed that TET2 mutations were frequently acquired at leukemic transformation [6 of 14 (43%)]. In contrast, ASXL1 mutations were almost always detected in both the MPN and AML clones from individual patients. One case was also observed where TET2 and ASXL1 mutations were found before the patient acquired a JAK2 mutation or developed clinical evidence of MPN. We conclude that mutations in TET2, ASXL1, and IDH1 are common in sAML derived from a preexisting MPN. Although TET2/ASXL1 mutations may precede acquisition of JAK2 mutations by the MPN clone, mutations in TET2, but not ASXL1, are commonly acquired at the time of leukemic transformation. Our findings argue that the mutational order of events in MPN and sAML varies in different patients, and that TET2 and ASXL1 mutations have distinct roles in MPN pathogenesis and leukemic transformation. Given the presence of sAML that have no preexisting JAK2/TET2/ASXL1/IDH1 mutations, our work indicates the existence of other mutations yet to be identified that are necessary for leukemic transformation. Cancer Res; 70(2); 447–52

Purpose We conducted a phase II study of pegylated interferon alfa-2a (PEG-IFN-α-2a) in patients with essential thrombocythemia (ET) and polycythemia vera (PV). Patients and Methods Seventy-nine patients (40 with PV and 39 with ET) have been treated. Median time from diagnosis to PEG-IFN-α-2a was 54 months in patients with PV and 33 months in patients with ET. Eighty-one percent of patients had received prior therapy. The first three patients received PEG-IFN-α-2a at 450 μg weekly. As a result of poor tolerance, this dose was decreased in a stepwise manner to a current starting dose of 90 μg weekly. Seventy-seven patients are evaluable and have been observed for a median of 21 months. Results The overall hematologic response rate was 80% in PV and 81% in ET (complete in 70% and 76% of patients, respectively). The JAK2V617F mutation was detected in 18 patients with ET and 38 patients with PV; sequential measurements by a pyrosequencing assay were available in 16 patients with ET and 35 patients with PV. The molecular response rate was 38% in ET and 54% in PV, being complete (undetectable JAK2V617F) in 6% and 14%, respectively. The JAK2V617F mutant allele burden continued to decrease with no clear evidence for a plateau. The tolerability of PEG-IFN-α-2a at 90 μg weekly was excellent. Conclusion PEG-IFN-α-2a resulted in remarkable clinical activity, high rates of molecular response, and acceptable toxicity in patients with advanced ET or PV. The ability of PEG-IFN-α-2a to induce complete molecular responses suggests selective targeting of the malignant clone.

Gain-of-function D816V point mutation within the kinase domain of the transmembrane receptor KIT is found in the great majority of patients with systemic mastocytosis (SM) and is attractive therapeutic target. Twenty patients with SM were enrolled during 2003-2005 in phase II clinical trial with imatinib mesylate (400mg daily), a KIT inhibitor. Median time on therapy was 9 months (range, 0.5-44+). Only one patient, with D816V KIT mutation-negative FIP1L1-PDGFRalpha-negative SM-HES, achieved complete remission (now lasting for 44 months). Six other patients reported symptomatic improvement, including two with D816V KIT mutation-positive SM (one reported improvement in diarrhea and the other in fatigue). Other patients had no benefit. Imatinib was relatively well tolerated. Our study confirms that imatinib therapy does not result in appreciable clinical activity in patients with D816V mutation-positive SM, but may result in a significant benefit in occasional patient with D816V mutation-negative SM.

Purpose To investigate the safety and efficacy of the combination of lenalidomide and prednisone in patients with myelofibrosis (MF). Patients and Methods Forty patients with MF were treated. Therapy consisted of lenalidomide 10 mg/d (5 mg/d if baseline platelet count < 100 × 109/L) on days 1 through 21 of a 28-day cycle for six cycles, in combination with prednisone 30 mg/d orally during cycle 1, 15 mg/d during cycle 2, and 15 mg/d every other day during cycle 3. Lenalidomide therapy was continued indefinitely in patients exhibiting clinical benefit. Results The median follow-up was 22 months (range, 6 to 27). Responses were recorded in 12 patients (30%) and are ongoing in 10 (25%). The median time to response was 12 weeks (range, 2 to 32). According to the International Working Group for Myelofibrosis Research and Treatment consensus criteria, three patients (7.5%) had partial response and nine patients (22.5%) had clinical improvement durable for a median of 18 months (range, 3.5 to 24+). Overall response rates were 30% for anemia and 42% for splenomegaly. Moreover, 10 of 11 assessable responders who started therapy with reticulin fibrosis grade 4 experienced reductions to at least a score of 2. All eight JAK2V617F–positive responders experienced a reduction of the baseline mutant allele burden, which was greater than 50% in four, including one of whom the mutation became undetectable. Grade 3 to 4 hematologic adverse events included neutropenia (58%), anemia (42%), and thrombocytopenia (13%). Conclusion The combination of lenalidomide and prednisone induces durable clinical, molecular, and pathologic responses in MF.

Polycythemia vera (PV) is a chronic myeloproliferative neoplasm (MPN) characterized by excessive production of red blood cells. Patients with PV are at a risk of thrombosis, bleeding, and transformation to myelofibrosis or acute myeloid leukemia. Therapy for PV is based on the use of phlebotomy, aspirin, and in high-risk patients, cytoreductive agents such as hydroxyurea. Anecdotal evidence suggests that imatinib mesylate, a selective tyrosine kinase inhibitor of ABL1, ARG, PDGFR, and KIT kinases has activity in PV. We conducted an open-label phase II clinical trial of imatinib at the standard dose of 400 mg daily in 24 patients with PV. The median duration of imatinib therapy was 5.1 months (range 0.2-86.4). Overall, 4 (17%) patients responded: one had a complete and three partial hematological response. The median time to response was 17.5 months (range 6-28), and the median duration of response was 17 months (range 9-68). No significant changes in JAK2(V617F) mutation burden were noted during imatinib therapy when compared with pretreatment values (P = 0.46). Therapy with imatinib was generally well tolerated. Our data indicate that imatinib has minimal clinical activity in PV.

In the World Health Organization classification, one major and four minor criteria are specified for the diagnosis of systemic mastocytosis. We report our experience using these criteria to diagnose systemic mastocytosis involving bone marrow. A total of 59 patients with clinically suspected systemic mastocytosis underwent comprehensive bone marrow examination, including immunophenotyping by immunohistochemistry and/or flow cytometry and molecular studies for KIT exon 17 mutations. Serum tryptase levels were also assessed. Of these 59, 53 (90%) patients met the diagnostic criteria for systemic mastocytosis. In these patients, multifocal dense infiltrates of mast cells, the major criterion, was observed in 36 (68%) patients. Atypical mast cell morphology was observed in 53 (100%), an aberrant immunophenotype was identified in 50 of 52 (96%), KIT mutation was present in 33 of 44 (75%), and an elevated serum tryptase (>20 ng/ml) was detected in 44 of 52 (85%). In the six patients in which bone marrow examination could not confirm systemic mastocytosis, one had systemic mastocytosis involving spleen, one patient had chronic idiopathic myelofibrosis, and four had no specific diagnosis, but systemic mastocytosis was still considered most likely. Of these six patients, atypical mast cell morphology was identified in five, aberrant immunophenotype in five, KIT mutation in two, and elevated serum tryptase in two. None of these cases met the major criteria. We conclude that the World Health Organization criteria are useful for the diagnosis of systemic mastocytosis in bone marrow specimens. The results also show the relative values of traditional morphologic criteria (ie, major criterion) and the results of ancillary testing (ie, minor criteria). However, as illustrated by the case of splenic systemic mastocytosis as well as the patient with chronic idiopathic myelofibrosis, the current World Health Organization system is neither completely sensitive nor specific for systemic mastocytosis.

Purpose: Molecular characterization of Philadelphia chromosome–negative (Ph−) chronic myeloproliferative disorders, such as systemic mastocytosis (SM), has provided a clear rationale for investigating novel targeted therapies. The tyrosine kinase (TK) inhibitor dasatinib is 325-fold more potent against Bcr-Abl TK than imatinib in vitro, significantly inhibiting wild-type KIT and platelet-derived growth factor receptor β TKs, and is active against cells carrying the mutant KIT-D816V gene. Experimental Design: In this phase 2, open-label study, the efficacy of dasatinib (140 mg/d) was investigated in 67 patients with various Ph− myeloid disorders, including SM (n = 33; 28 KIT-D816V positive). Results: The overall response rate to dasatinib in patients with SM was 33%. Only two patients, one with SM-myelofibrosis and one with SM-chronic eosinophilic leukemia, achieved complete response (elimination of mastocytosis) lasting for 5 and 16 months, respectively. Both patients were negative for KIT-D816V mutation, had low tryptase levels, abnormal WBC counts, and anemia, and had failed prior therapy with erythropoietin. Additional nine SM patients had symptomatic response, lasting 3 to 18+ months. Complete responses were achieved in two other patients (acute myeloid leukemia and hypereosinophilic syndrome). No responses were observed among patients with myelodysplastic syndromes and primary myelofibrosis. The majority of adverse events were grade 1/2. Conclusion: These data show that dasatinib therapy may benefit a selected group of SM patients, primarily by improving their symptoms, but it does not eliminate the disease in the patients with KIT-D816V mutation.

Chronic lymphocytic leukemia (CLL) and myeloproliferative neoplasms (MPN) may occur concomitantly. However, little is known about the pathobiological characteristics and interaction between the neoplastic clones in these rare cases of coinciding malignancies. We retrospectively examined the clinical and biological characteristics of 13 patients with concomitant CLL and MPN--eight primary myelofibrosis (PMF), three essential thrombocytosis (ET), and two polycythemia vera (PV)--who presented to our institution between 1998 and 2014, and tested all patients for MPN-specific aberrations, such as JAK2, MPL and CALR mutations. Along with epidemiological and molecular characterization of this rare condition, we found that JAK2 mutation can be detected 9 years prior to PMF diagnosis, suggesting that PMF clinical phenotype may require several years to develop and CLL/MPN clinical co-occurrence might be sustained by common molecular events. Some features of these patients suggest that pathobiologies of these diseases might be intertwined.

Idiopathic hypereosinophilic syndrome (HES) is a myeloproliferative disorder characterized by tissue involvement and organ dysfunction due to abnormal eosinophil proliferation. In a subset of patients, this is caused by the FIP1L1-PDGFR-alpha fusion tyrosine kinase. Cumulative evidence indicates that the Bcr-Abl tyrosine kinase inhibitor imatinib mesylate (Gleevec) is active for the treatment of patients with HES, particularly those expressing the FIP1L1-PDGFR-alpha oncoprotein. The novel tyrosine kinase inhibitor AMN107 was initially developed as a potent Bcr-Abl inhibitor based on the molecular structure of imatinib. We tested the in vitro efficacy of imatinib and AMN107 in the EOL-1 cell line and in cells from a patient with HES harboring the FIP1L1-PDGFR-alpha fusion kinase. AMN107 was as potent as imatinib in inducing apoptosis and inhibiting proliferation of EOL-1 cells, with IC(50) values of 0.54 and 0.20 nM, respectively. In addition, both drugs inhibited the phosphorylation of PDGFR-alpha tyrosine kinase with equivalent efficacy. We conclude that AMN107 and imatinib are active and equipotent against cells expressing the FIP1L1-PDGFR-alpha fusion gene.

Adaphostin is a tyrphostin that was designed to inhibit Bcr/Abl tyrosine kinase by altering the binding site of peptide substrates rather than that of adenosine triphosphate, a known mechanism of imatinib mesylate (IM). However, it has been shown that adaphostin-mediated cytotoxicity is dependent on oxidant production and does not require Bcr/Abl. We have tested adaphostin against both Philadelphia chromosome (Ph)-positive (K562, KBM5, KBM5-R [IM resistant KBM5], KBM7, and KBM7-R [IM-resistant KBM7]) and Ph-negative (OCI/AML2 and OCI/AML3) cells, and against cells from patients with chronic myeloid leukemia (CML) and acute myeloid leukemia (AML). Adaphostin significantly inhibited growth of all cell lines (50% inhibition of cell proliferation [IC50] 0.5-1 microM) except K562 (IC50 13 microM). Ph-positive IM-resistant cell lines showed significant cross resistance to adaphostin. Simultaneous or sequential treatment with adaphostin and IM did not exert a synergistic effect in any KBM line. Adaphostin induced superoxide and apoptosis in a dose-dependent and time-dependent fashion in both Ph-positive and Ph-negative cells. Adaphostin selectively inhibited colony growth of cells from CML (IM-sensitive and IM-resistant) and AML patients. Analysis of tyrosine phosphorylated proteins after treatment with adaphostin revealed alternate effects in different cells consistent with the modulation of multiple targets. In conclusion, adaphostin showed significant and selective activity against CML and AML cells and its development for clinical testing is warranted.

Patients with myeloproliferative neoplasms (MPNs) are at significant, cumulative risk of leukemic transformation to acute myeloid leukemia (AML), which is associated with adverse clinical outcome and resistance to standard AML therapies. We performed genomic profiling of post-MPN AML samples; these studies demonstrate somatic tumor protein 53 (TP53) mutations are common in JAK2V617F-mutant, post-MPN AML but not in chronic-phase MPN and lead to clonal dominance of JAK2V617F/TP53-mutant leukemic cells. Consistent with these data, expression of JAK2V617F combined with Tp53 loss led to fully penetrant AML in vivo. JAK2V617F-mutant, Tp53-deficient AML was characterized by an expanded megakaryocyte erythroid progenitor population that was able to propagate the disease in secondary recipients. In vitro studies revealed that post-MPN AML cells were sensitive to decitabine, the JAK1/2 inhibitor ruxolitinib, or the heat shock protein 90 inhibitor 8-(6-iodobenzo[d][1.3]dioxol-5-ylthio)-9-(3-(isopropylamino)propyl)-9H-purine-6-amine (PU-H71). Treatment with ruxolitinib or PU-H71 improved survival of mice engrafted with JAK2V617F-mutant, Tp53-deficient AML, demonstrating therapeutic efficacy for these targeted therapies and providing a rationale for testing these therapies in post-MPN AML.

Summary. The cell surface proteins CD20 and CD52 differ significantly in their structures and are expressed on the majority of B cells. Both circulating CD20 (cCD20) and circulating CD52 (cCD52) have been recently documented in patients with chronic lymphocytic leukaemia. A retrospective study to establish whether cCD20 and/or cCD52 were detectable in patients with lymphoma, and the clinical associations of these soluble antigens if detected, was conducted. cCD20 and cCD52 levels were analysed in a cohort of 65 patients with non-Hodgkin's lymphoma (NHL) and 37 with Hodgkin's disease (HD). Patients with NHL had elevated pretherapy levels of cCD20 and cCD52 compared with normal individuals. Patients with HD had significantly lower than normal pretherapy levels of both cCD20 and cCD52. cCD20 levels were marginally elevated post-therapy in NHL patients while in patients with HD, cCD20 levels remained significantly lower than normal after therapy. Serum cCD52 levels became significantly lower than normal post-therapy in NHL patients, and remained significantly lower than normal in HD patients. No predictive effects were found for pretherapy or post-therapy levels of cCD52 on survival for either cohort of patients. Post-therapy cCD20 levels independently highly correlated with survival in patients with NHL. Prospective evaluation will be required to establish if cCD20 and cCD52 may be used as biomarkers in the diagnosis, prognostic categorization, and monitoring of the clinical course in patients with lymphoma. The clinical significance of circulating antigen in patients receiving monoclonal antibody therapy directed against CD20 and/or CD52 warrants study.

The genetic events involved in the transformation of chronic lymphocytic leukemia (CLL) to Richter's syndrome (RS) are poorly understood. Frequently large cells are seen in the bone marrows of patients with CLL and evidence of RS. Using a laser-capture microdissection we analyzed small and large leukemic bone marrow cells from 19 patients with RS for loss of heterozygosity (LOH) on chromosome 11 (D11S2179 at the ATM gene), 17 (D17S938 and D17S1852 at the TP53 site), and 20 (Plc1, D20S96, D20S110, and D20S119). Megakaryocytes were also isolated and used as a control for normal cells. Four of 15 (27.7%) informative cases showed LOH in small cells in the ATM gene while seven (46.7%) showed LOH in large cells. Six of 15 (40%) informative cases had LOH in chromosome 17 in small cells, and eight (53%) showed LOH in large cells. Eleven of 19 informative cases (61.1%) showed LOH in chromosome 20 in large cells, and eight (42.1%) showed LOH in small cells. RS cases with LOH at chromosome 20 were associated with marginally shorter survival rates (P = 0.08). Our data suggest that there are significant molecular differences between large and small cells in patients with CLL. Further analysis of the genes on these chromosomes may provide new insight into our understanding of the transformation of small CLL cells to large (Richter) cells.

Background: Deletions or structural abnormalities in chromosomes 11 and 13 have been shown to be important in predicting clinical behavior in patients with multiple myeloma (MM). However, cytogenetic analysis in MM is frequently difficult because of poor yield of informative metaphases and the disease is frequently patchy, which complicates fluorescent in situ hybridization studies. Objectives: The purpose of this study was to explore the potential of using peripheral plasma DNA for the detection of loss of heterozygosity (LOH) in chromosomes 11 and 13 in patients with MM. Methods: Peripheral blood (PB) plasma of 81 patients with MM, was used as a source of DNA for the detection of LOH at chromosomes 13q14 (D13S319 and D13AFMaw301wb5), and 11q21 (D11S2179) using polymerase chain reaction. Results: Only 62 of the studied patients were informative for the two 13q microsatellite markers and 16 (26%) of these patients showed LOH. Only seven (11%) of 61 patients with informative D11S2179 microsatellite maker showed LOH. Purified plasma cells (PCs) from six bone marrow (BM) samples using anti-CD138-coated magnetic beads showed identical results to those detected in DNA isolated from PB plasma. Three patients with LOH underwent autologous BM transplantation, and two of three reverted to a normal state (no LOH) after transplantation. Seven of the patients with 13q LOH in PB plasma had

BACKGROUND CD31 (platelet endothelial cell adhesion molecule-1 [PECAM-1]) is the ligand for CD38, a transmembrane glycoprotein that is expressed on the surface of leukemic cells in many patients with B-cell chronic lymphocytic leukemia (B-CLL). In a previous study, the authors showed that CD38 expression was correlated with a poor prognosis in patients with B-CLL. In the current study, blood samples from patients with B-CLL were examined to identify CD31 surface marker expression, and CD31 expression was correlated with several other known prognostic variables, including CD38. METHODS Using flow cytometry, peripheral blood samples from 120 patients with B-CLL were analyzed for CD31 and CD38 expression on CD19 positive leukemic B cells. RESULTS Thirteen of 120 patients (11%) had CD31 expression on < 20% of their B cells, and the remaining patients had various levels of CD31 expression. The median expression of CD31 was 76% of leukemic, CD19 positive cells. Levels of CD31 expression were not correlated with survival outcomes or with any of the known prognostic parameters when all patients were considered. Patients who had high CD38 expression (≥ 20%), as expected, had significantly shorter survival (P = 0.001) compared with patients who had low CD38 expression (< 20%). However, in patients with low CD38 expression, a subgroup with low CD31 expression (< 76%) had significantly longer survival compared with the survival for the entire group (P = 0.0001). Moreover, the survival pattern of patients with low CD38 expression and high CD31 expression was not significantly different from the survival pattern seen in patients with high CD38 expression. CONCLUSIONS CD31 expression further defined a subgroup of patients with B-CLL who had a different survival outcome. Defining the interaction between CD31 expression and CD38 expression in patients with CLL will require further exploration. Cancer 2003;97:1914–9. © 2003 American Cancer Society. DOI 10.1002/cncr.11264

CD20 is a 33- to 36-kDa transmembrane phosphoprotein involved in the activation, proliferation, and differentiation of B lymphocytes. The predicted amino acid sequence of the CD20 suggests 4 transmembrane-spanning regions with both N- and C-termini located in the cytoplasm. We demonstrate herein that significant levels of circulating CD20 (cCD20) can be detected in the plasma of patients with chronic lymphocytic leukemia (CLL) and that cCD20 interferes with the binding of rituximab, a humanized anti-CD20 monoclonal antibody, to CLL cells. An enzyme-linked immunosorbent assay (ELISA) was developed to measure circulating cCD20 levels in the plasma. We measured cCD20 levels in the plasma of 180 patients with CLL and correlated these levels with clinical characteristics and outcome. Circulating CD20 levels correlated positively with β2-microglobulin level (p = .006) and percentage of CD38+ cells (p = .03) and negatively with platelet count (p = .004) and hemoglobin level (p = .02). Patients with advanced Rai (III/IV) or Binet (C) stage disease had significantly higher levels of cCD20 than did patients with earlier-stage disease (P = .01 and P = .006, respectively). There was no correlation between cCD20 level and age, lymphocyte count, or white blood cell count. Using a recursive classification method, we found that patients with a cCD20 level more than 1875 nM/L had significantly shorter survival than those with cCD20 1875 nM/L or below (P = .01). The prognostic value of cCD20 was independent of Rai staging or hemoglobin level. Prospective evaluation is indicated to establish whether rituximab dosing should be adjusted according to cCD20 levels.