A Calreticulin Neoepitope-Directed Monoclonal Antibody Can Overcome JAK Inhibitor Resistance and Block TPO-Independent Megakaryocyte Differentation

Abstract Introduction: Mutations within the gene encoding calreticulin (CALR) are the second most common genetic aberration associated with primary myelofibrosis (PMF), observed in 70% of non-JAK2 V617F cases. Importantly, patients with CALR mutations do not effectively respond to JAK inhibitor therapy and no mutation specific therapy is currently in use. Virtually all CALR mutations identified in PMF are small insertions or deletions clustered within exon 9 leading to a neo-epitope peptide sequence which is thought to directly or indirectly activate the thrombopoietin receptor (TpoR) by a poorly defined mechanism. Here we engineered a neo-epitope specific monoclonal antobody that has striking biological activity against ruxolitinib persistent cells. Methods TF-1 TpoR cells expressing TpoR were supplemented with 20 ng/mL of TPO. Rats were immunised with a CALR mutant peptide coupled to KLH. Serum from the immunised rats was screened by enzyme linked immunoassay, to verify a strong titre to the peptide immunogen. Primary PMF CD34+ cells were cultured in StemCell Pro with human SCF, IL-6 and IL-9. NSG mice were used to for engraftment studies after 150 cGy irradiation. Results: We engineered a panel of rat monoclonal antibodies after immunization with a 30 amino acid peptide corresponding to the C-terminal mutant CALR neoepitope sequence with an extra cysteine residue. Clone 4D7 showed superior activity of detecting mutant but not wild type CALR protein with a binding affinity of 13.5 pM and dissociation constant of 1.53 nM as measured by I 125-Scatchard. Treatment with 4D7 resulted in a significant (5-7-fold) increase in the amount of full-length mutant CALR protein in conditioned media. 4D7 inhibited Tpo-independent cell growth over 6 days in TF-1 cells expressing MPL and mutant CALR at 2, 10 and 20 µg. 4D7 blocked constitutive factor-independent phospho-STAT5 and phospho-ERK after incubation exclusively in mutant CALR cells but not in TF-1 cells expressing TpoR alone and increased the sub-G 0 fraction was observed compared to IgG control (P = 0.001, n = 3 independent experiments) consistent with induction of an apoptotic response. We tested activity in purified primary CD34+ cells obtained from patients with CALR mutant myelofibrosis using two orthogonal assays: - (i) Tpo-independent megakaryocyte differentiation in liquid culture and (ii) Tpo-independent megakaryocyte colony formation on a collagen-based medium. 4 out of 4 patient samples that displayed robust Tpo-independent growth of CD41+CD61+ megakaryocyte progenitors showed inhibition by 4D7 of at least 50%. Similarly, we saw dramatic reduction in the absolute numbers of primary Tpo-independent megakaryocyte colonies cultured on collagen (colony-forming unit-mega) treated with 4D7 in multiple patient samples (decrease of 46%, P = 0.0001, Student's t-test, n = 4 independent patient samples) Importantly, secretion of mutant CALR protein was neither upregulated nor downregulated by ruxolitinib, indicating ruxolitinib is unlikely to alter mutant CALR trafficking in patients. 4D7 had strong inhibitory activity on cells that were resistant to ruxolitinib, in both liquid culture at 96 hours or colony formation. To test whether 4D7 could block mutant CALR-dependent proliferation in vivo, we developed two distinct xenograft models, a bone marrow engraftment model, which measures mutant CALR dependent proliferation in the bone marrow microenvironment, and a chloroma model, which mimics extravascular infiltration of mutant CALR leukaemia, by injection of TPO-independent TF-1 cells in NSG mice. In the bone marrow engraftment model 4D7 treatment (12 mg/kg twice weekly via intraperitoneal injection) lowered peripheral blood engraftment of human CD33 myeloid cells at 3 weeks, bone marrow engraftment and significantly prolonged survival compared to IgG control (P=0.004, HR=0.2). In the chloroma model, 4D7 treatment resulted in significant decrease in tumour growth measured at 3 weeks (P<0.01) and improved overall survival (P=0.02, HR=0.07) compared to IgG control Conclusion: Together, these results suggest an immunotherapeutic approach may have clinical utility CALR-driven myeloproliferative neoplasms and CALR mutant acute myeloid leukaemia, as well as activity in CALR mutant patients that develop resistance/persistence to ruxolitinib. Disclosures Ross: Bristol Myers Squib: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Keros Therapeutics: Consultancy, Honoraria. Reinisch: Celgene: Research Funding; Pfizer: Consultancy.

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Inhibition of Chronic Myeloid Leukemia Stem Cells by the Combination of the Hedgehog Pathway Inhibitor LDE225 with Nilotinib

Blood ◽

10.1182/blood.v116.21.514.514 ◽

2010 ◽

Vol 116 (21) ◽

pp. 514-514 ◽

Cited By ~ 6

Author(s):

Bin Zhang ◽

David Irvine ◽

Yin Wei Ho ◽

Silvia Buonamici ◽

Paul Manley ◽

...

Keyword(s):

Stem Cells ◽

Board Of Directors ◽

Colony Formation ◽

Research Funding ◽

Leukemia Stem Cells ◽

Advisory Committees ◽

Nsg Mice ◽

Cd34 Cells ◽

Hh Signaling

Abstract Abstract 514 Background: Tyrosine kinase inhibitors (TKI), although effective in inducing remissions and improving survival in CML patients, fail to eliminate leukemia stem cells (LSC), which remain a potential source of relapse on stopping treatment. Additional strategies to enhance elimination of LSC in TKI-treated CML patients are required. The Hedgehog (Hh) pathway, important for developmental hematopoiesis, has been shown to be activated in BCR-ABL-expressing LSC, in association with upregulation of Smoothened (SMO), and contributes to maintenance of BCR-ABL+ LSC. However the role of Hh signaling in chronic phase (CP) CML LSC is not clear. LDE225 (LDE, Novartis Pharma) is a small molecule SMO antagonist which is being clinically evaluated in patients with solid tumors. We have reported that LDE does not significantly affect proliferation and apoptosis of primary CP CML CD34+ cells, or reduce colony growth in CFC assays, but results in significant reduction in CML CFC replating efficiency and secondary colony formation. Treatment with LDE + Nilotinib resulted in significant reduction in colony formation from CD34+ CML cells in LTCIC assays compared to Nilotinib alone or untreated controls. These observations suggest that LDE may preferentially inhibit growth of primitive CML progenitors and progenitor self-renewal. We therefore further investigated the effect of LDE on growth of primitive CML LSC in vivo. Methods and Results: 1) CP CML CD34+ cells were treated with LDE (10nM), Nilotinib (5μ M) or LDE + Nilotinib for 72 hours followed by transplantation into NOD-SCID γ-chain- (NSG) mice. Treatment with LDE + Nilotinib resulted in reduced engraftment of CML CD45+ cells (p=0.06) and CD34+ cells (p=0.02) compared with controls, and significantly reduced engraftment of CML cells with CFC capacity (p=0.005). In contrast LDE or Nilotinib alone did not reduce CML cell engraftment in the bone marrow (BM) compared with untreated controls. LDE, Nilotinib, or LDE + Nilotinib treatment did not significantly inhibit engraftment of normal human CD34+ cells in NSG mice compared to controls. 2) We also used the transgenic Scl-tTa-BCR-ABL mouse model of CP CML to investigate the effect of in vivo treatment with LDE on CML LSC. BM cells from GFP-SCL-tTA/BCR-ABL mice were transplanted into wild type congenic recipients to establish a cohort of mice with CML-like disease. Recipient mice developed CML-like disease 3–4 weeks after transplantation. Transplanted CML cells were identifiable through GFP expression. Mice were treated with LDE225 (80mg/kg/d by gavage), Nilotinib (50 mg/kg/d by gavage), LDE + Nilotinib, or vehicle alone (control) for 3 weeks. Treatment with Nilotinib, LDE, and LDE + Nilotinib resulted in normalization of WBC and neutrophil counts in peripheral blood. LDE + Nilotinib treatment significantly reduced the number of splenic long term hematopoietic stem cells (LT-HSC, Lin-Sca-1+Kit+Flt3-CD150+CD48-, p<0.01) and granulocyte-macrophage progenitors (GMP) compared to controls, but did not significantly alter LT-HSC numbers in the BM. LDE alone reduced splenic LT-HSC but not GMP, whereas Nilotinib alone did not reduce LT-HSC numbers in spleen or BM but significantly reduced splenic GMP numbers. The mechanisms underlying enhanced targeting of LSC in the spleen compared to the BM are not clear but could reflect greater dependence on Hh signaling in the context of the splenic microenvironment and/or relocalization of LDE treated LT-HSC to BM. Experiments in which BM and spleen cells from treated mice were transplanted into secondary recipients to determine functional stem cell capacity of remaining LT-HSC are ongoing. Importantly mice treated with LDE + Nilotinib demonstrated enhanced survival on follow up after discontinuation of treatment compared with control mice or mice treated with LDE or Nilotinib alone. Conclusions: We conclude that LDE225 can target LSC from CP CML patients and in a transgenic BCR-ABL model of CP CML, and that LDE + Nilotinib treatment may represent a promising strategy to enhance elimination of residual LSC in TKI-treated CML patients. Disclosures: Buonamici: Novartis: Employment. Manley:Novartis: Employment. Holyoake:Novartis: Consultancy, Research Funding. Copland:Novartis Pharma: Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees. Bhatia:Novartis: Consultancy, Honoraria.

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Phase 2 Trial of PRM-151, an Anti-Fibrotic Agent, in Patients with Myelofibrosis: Stage 1 Results

Blood ◽

10.1182/blood.v124.21.713.713 ◽

2014 ◽

Vol 124 (21) ◽

pp. 713-713 ◽

Cited By ~ 29

Author(s):

Srdan Verstovsek ◽

Ruben A. Mesa ◽

Lynda M Foltz ◽

Vikas Gupta ◽

John O Mascarenhas ◽

...

Keyword(s):

Bone Marrow ◽

Board Of Directors ◽

Clinical Benefit ◽

Research Funding ◽

Overall Response Rate ◽

Response Rate ◽

Jak Inhibitor ◽

Advisory Committees ◽

Grade 3 ◽

Stage 1

Abstract PRM-151 (PRM) is a recombinant form of Pentraxin-2, an endogenous human protein that acts at sites of tissue damage, inducing macrophage differentiation to prevent and reverse fibrosis. PRM has broad anti-fibrotic activity in multiple preclinical models of established fibrotic diseases and no dose limiting toxicities in phase 1 trials. Myelofibrosis (MF: primary (PMF), post-essential thrombocythemia (post-ET MF), and post polycythemia vera (Post PV MF)) is a myeloid malignancy characterized by progressive bone marrow (BM) fibrosis with resultant anemia, abnormal platelet and leukocyte counts, extramedullary hematopoiesis, and a well-defined symptom complex. This study investigated the potential of PRM in MF to reduce BM fibrosis and to improve key disease features including abnormal blood counts, symptoms, and splenomegaly. MF patients (pts) with Dynamic International Prognostic Scoring System (DIPSS) intermediate-1, intermediate-2, or high-risk disease and grade ≥ 2 BM fibrosis, either on no current therapy or on a stable dose of ruxolitinib (RUX) for ≥ 12 weeks and no improvement in spleen for ≥ 4 weeks, were eligible for stage 1 of this open-label adaptive trial. Assignment to one of the 4 treatment arms was per investigator and pt choice: PRM 10 mg/kg IV 1-hour infusion days 1, 3, 5, then weekly (QW) or every 4 weeks (Q4W), alone or with RUX, for 24 weeks. Primary endpoint was overall response rate by IWG-MRT (symptoms by MPN-SAF Total Symptom Score (TSS), spleen by palpation) and/or decrease in BM fibrosis by ≥ 1 grade with otherwise stable disease. BM biopsies were obtained at baseline, 3 and 6 months, and were evaluated centrally by two blinded hematopathologists. Pts with clinical benefit were allowed to continue treatment in an extension. At least one response in any arm was required for that regimen to be evaluated in Stage 2. Twenty seven pts were enrolled: 8 PRM QW, 7 PRM Q4W, 6 PRM QW + RUX, 6 PRM Q4W + RUX. Median age 67 years (52-85); 70% DIPSS Int-2 or High Risk; 52% PMF, 15% post-ET MF, 33% post-PV MF; 63% grade 3 BM fibrosis, Hemoglobin (Hgb) < 100 g/L in 56% and < 85 g/L in 26%, platelet count (PLT)< 100 x 109/L in 52% and < 25 x 109/L in 30%; 22% were JAK inhibitor-naive and 52% had received a prior JAK inhibitor (not including ongoing RUX). Twenty pts completed 24 weeks of therapy; 18 continued extension treatment. PRM-151 was well-tolerated alone and with RUX; most adverse events (AEs) were Grade 1/2 and unrelated, with 3 Grade 3 possibly related AEs and 5 possibly related serious AEs. Nine of 26 evaluable pts responded, for an overall response rate (ORR) of 35%, with 4 IWG symptom clinical improvements (CI) and 6 BM fibrosis responses (Table 1), with ≥ 1 response in each arm. One pt had a CI and BM response. Reduction in BM fibrosis was associated with normal erythroid microarchitecture, normal or decreased myeloid:erythroid ratio, and fewer paratrabecular megakaryocytes, all potential surrogates of improved bone marrow microenvironment. IWG stable disease was observed in 77% of pts, with trends of clinical benefit in Hgb, PLT, peripheral blood blasts, spleen, and symptoms (Table 2). In 14 patients (54%), all parameters were stable or improved. Conclusion: PRM-151 was well-tolerated in patients with advanced MF, with no evidence of drug-related myelosuppression and encouraging trends in both clinical and histologic aspects of the disease. Reduction in BM fibrosis, stable to improved hematologic parameters, symptom responses, and stable to reduced spleen size support further development of PRM-151 in MF. Table 1 Two additional subjects had decrease in bone marrow fibrosis but progressive disease. Number of Patients BM Fibrosis Grade at Last Study Timepoint 3 2 1 BM Fibrosis Grade at Baseline 3 8 3 1 2 1 4 2 Abstract 713. Table 2 Outcome Parameter Denominator (n) Clinical Benefit Pts with Improvement (n/%) ORR (primary endpoint) All evaluable pts (26) IWG-MRT CI AND/OR reduction in BM fibrosis by ≥ 1 grade 9 (35%) Hgb Hgb < 100 g/L (15) ≥10 g/L increase from baseline AND no transfusions or 50% reduction in transfusions if transfusion dependent 6 (40%) PLT PLT < 100 x 109/L (13) > 100 x 109/L AND increase of ≥20 x 109/L ; increase of ≥20 x 109/L if baseline < 50, AND/OR increase of ≥ 10 x 109/L with discontinuation of transfusions 8 (62%) Blasts ≥ 1% peripheral blasts (14) No peripheral blasts 3 (21%) Symptoms All evaluable pts (26) ≥ 25% reduction in TSS ≥ 12 weeks 10 (38%) Spleen Palpable spleen (19) ≥ 25% decrease ≥ 4 weeks AND any decrease ≥ 12 weeks 5 (26%) Disclosures Verstovsek: Incyte: Research Funding; Astrazeneca: Research Funding; Lilly Oncology: Research Funding; Roche: Research Funding; Geron: Research Funding; NS Pharma: Research Funding; Bristol Myers Squibb: Research Funding; Novartis: Research Funding; Celgene: Research Funding; Gilead: Research Funding; Seattle Genetics: Research Funding; Promedior: Research Funding; Cell Therapeutics: Research Funding. Mesa:Incyte, CTI, NS pharma, Gilead, Celgene: Research Funding; Promedior: Research Funding. Foltz:Janssen: Consultancy; Promedior: Research Funding; Gilead: Research Funding; Incyte: Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Gupta:Incyte Corporation: Consultancy, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Promedior: Research Funding. Mascarenhas:Novartis Pharmaceuticals Corporation: Research Funding; Incyte Corporation: Consultancy, Research Funding; Promedior: Research Funding. Ritchie:Celgene, Incyte: Speakers Bureau; Promedior: Research Funding. Hoffman:Geron: Consultancy, Membership on an entity's Board of Directors or advisory committees; All Cells LLC: Consultancy, Membership on an entity's Board of Directors or advisory committees; Promedior: Research Funding. Pozdnyakova:Sanofi: Consultancy; Incyte: Consultancy; Promedior: Consultancy. Hasserjian:Sanofi: Consultancy; Incyte: Consultancy; Promedior: Consultancy. Trehu:Promedior: Employment, Equity Ownership. Kantarjian:ARIAD, Pfizer, Amgen: Research Funding. Gotlib:Novartis: Research Funding, Travel Reimbursement, Travel Reimbursement Other; Sanofi: Research Funding; Gilead: Research Funding; Incyte: Consultancy, Honoraria, Research Funding, Travel Reimbursement Other; Promedior: Research Funding.

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RON Kinase Is a Novel Therapeutic Target for Philadelphia-Negative Myeloproliferative Neoplasms

Blood ◽

10.1182/blood-2021-148192 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1462-1462

Author(s):

Lindsay Meg Gurska ◽

Rachel Okabe ◽

Meng Maxine Tong ◽

Daniel Choi ◽

Kristina Ames ◽

...

Keyword(s):

Bone Marrow ◽

Board Of Directors ◽

Research Funding ◽

Myeloproliferative Neoplasms ◽

Marrow Transplant ◽

Jak Inhibitor ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Stat Signaling ◽

Genetic Deletion

Abstract The Philadelphia-chromosome negative myeloproliferative neoplasms (MPNs), including polycythemia vera (PV), essential thrombocytosis (ET), and primary myelofibrosis (PMF), are clonal hematopoietic stem cell disorders characterized by the proliferation of one or more myeloid lineage compartments. Activation of JAK/STAT signaling is a major driver of all Ph-negative MPNs. During disease progression, MPN patients experience increased pro-inflammatory cytokine secretion, leading to remodeling of the bone marrow microenvironment and subsequent fibrosis. The JAK inhibitor ruxolitinib is an approved targeted therapy for MPN patients and has shown promise in its ability to reduce splenomegaly and the cytokine storm observed in patients. However, JAK inhibitors alone are not sufficient to reduce bone marrow fibrosis or to eliminate the JAK2-mutated clone. Furthermore, JAK inhibitor persistence, or reactivation of JAK/STAT signaling upon chronic JAK inhibitor treatment, has been observed in both MPN mouse models and MPN patients. Therefore, there is an urgent need for new treatment options in MPN. The tyrosine kinase RON, a member of the MET kinase family, has well-characterized roles in erythroblast proliferation and pro-inflammatory cytokine production. RON can be phosphorylated by JAK2 to stimulate erythroblast proliferation. However, the role of RON in MPN pathogenesis is unknown. We found that the ALK/MET/RON/ROS1 inhibitor crizotinib inhibited colony formation by MPN patient CD34+ cells, regardless of their disease subtype, mutation status, or JAK2 inhibitor treatment history (Figure 1A). To determine whether this is due to inhibition of the JAK/STAT signaling pathway, we performed phospho-flow cytometry of STAT3 and STAT5 in myelofibrosis patient erythroblasts treated with crizotinib ex vivo as well as Western blot analysis in the JAK2-mutated cell lines SET2 and HEL. We found that crizotinib inhibits the phosphorylation of JAK2, STAT3, and STAT5 (Figure 1B). Since crizotinib has not been reported to directly inhibit JAK2, we asked whether these effects of crizotinib in MPN cells could be explained by RON inhibition. Consistent with this hypothesis, we observed that shRNA knockdown of multiple RON isoforms also decreases the phosphorylation of JAK2, STAT5, and STAT3 in HEL cells (Figure 1C-D). To determine whether crizotinib can alter the MPN disease course in vivo, we tested crizotinib by oral gavage in the MPLW515L bone marrow transplant murine model of myelofibrosis at 100mg/kg daily for 2 weeks. We showed that crizotinib decreased the disease burden of MPL-W515L mice, as evidenced by decreased spleen and liver weights (Figure 1E). To determine the effects of RON genetic deletion on MPN pathogenesis, we tested whether genetic deletion of Stk (mouse gene for RON) impairs disease progression in the JAK2V617F bone marrow transplant MPN model by transplanting Stk-/- c-Kit+ bone marrow cells transduced with the JAK2V617F-GFP retrovirus into lethally irradiated recipients. We observed a significant delay in disease onset in Stk-/- transplant recipients compared to WT controls (Figure 1F). However, we found that Stk-/- mice have normal numbers of hematopoietic stem and progenitor cells, and normal bone marrow myeloid colony forming capacity, suggesting that RON is a safe therapeutic target. To determine whether RON plays a role in the JAK inhibitor persistence phenotype, we generated persistent cells by treating SET2 cells with increasing doses of ruxolitinib over 8 weeks, and confirmed persistent proliferation and JAK/STAT activation. Interestingly, we found that RON phosphorylation is enhanced in JAK inhibitor persistent cells, and that dual inhibition of RON and JAK2 overcomes JAK inhibitor persistence in SET2 cells (Figure 1G-H), suggesting that RON may potentiate the JAK2 persistence phenotype in response to ruxolitinib. Importantly, we showed by immunoprecipitation that phospho-RON and phospho-JAK2 physically interact in JAK inhibitor persistent SET2 cells, and that this interaction is disrupted by crizotinib (Figure 1I). In summary, our data demonstrate that RON kinase is a novel mediator of JAK/STAT signaling in MPNs, and that it plays a particularly important role in JAK inhibitor persistence. Our work suggests that therapeutic strategies to inhibit RON, such as crizotinib, should be investigated in MPN patients. Figure 1 Figure 1. Disclosures Halmos: Guardant Health: Membership on an entity's Board of Directors or advisory committees; Apollomics: Membership on an entity's Board of Directors or advisory committees; TPT: Membership on an entity's Board of Directors or advisory committees; Eli-Lilly: Research Funding; Advaxis: Research Funding; Blueprint: Research Funding; Elevation: Research Funding; Mirati: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer-Ingelheim: Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Astra-Zeneca: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding. Gritsman: iOnctura: Research Funding.

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Decreased Expression of CD62L and CD54 Correlates with Poor Cytogenetic Risk Group In Myelodysplastic Syndromes

Blood ◽

10.1182/blood.v116.21.2915.2915 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2915-2915

Author(s):

Canan Alhan ◽

Theresia M. Westers ◽

Claudia Cali ◽

Floortje L. Kessler ◽

Monique Terwijn ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Healthy Volunteers ◽

Board Of Directors ◽

Myelodysplastic Syndromes ◽

Research Funding ◽

Risk Category ◽

Advisory Committees ◽

Cd34 Cells

Abstract Abstract 2915 Interactions in the bone marrow (BM) between haematopoietic progenitor cells (HPC) and the BM micro environment are important for the regulation of cell adhesion, proliferation, differentiation and survival. Expression of both CD62L (L-selectin) and CD54 (ICAM-1) on HPC demonstrated to play a role in signal transduction routes for proliferation and growth regulation. Especially CD54 is involved in uncontrolled proliferation and block of apoptosis. Previously, it was described that decreased expression of CD62L in acute myeloid leukemia (AML) was associated with a poor cytogenetic risk profile and an adverse clinical outcome (Graf M et al, Eur J Haematol 2003) Myelodysplastic syndromes are a group of clonal HPC disorders characterized by ineffective hematopoiesis and a propensity to evolve into AML. The International Prognostic Scoring System (IPSS) provides information on both survival and risk of development of an AML. The purpose of our study was to evaluate CD62L and CD54 expression on CD34+ cells in MDS patients by flow cytometry and to assess the value of a CD62L/CD54 ratio for prognostication. Bone marrow samples of 30 newly diagnosed MDS patients (3 RA(RS)/18 RCMD(RS), the <5% blasts group; 5 RAEB-1, 4 RAEB-2, the >5% blasts group), 16 AML patients with prior MDS and 26 healthy volunteers were analyzed for CD62L and CD54 expression on CD34+ cells by using flow cytometry. An adhesion index was calculated as a ratio of the percentage and MFI of CD62L and CD54 positive cells (as was reported by Buccisano et al, Eur J Haematol 2007). The CD62L/CD54 ratio was significantly decreased in MDS with <5% blasts (median 79.09 p<0.0001) as compared to healthy volunteers (median 480.4) and even more decreased in high risk MDS (median 14.67 p<0.0001 and p=0.001 as compared to healthy volunteers and MDS with <5% blasts, respectively) and AML with prior MDS (median 12.54, p<0.0001 and p=0.009 as compared to healthy volunteers and MDS with <5% blasts, respectively). The MDS patients were assigned to the good, intermediate or poor IPSS cytogenetic risk category. Cytogenetics was available for 22 MDS patients. The CD62L/CD54 ratio was significantly lower in the cytogenetic poor risk category compared with the good risk category (median 5.4 and median 70.79 respectively, p=0.018). Moreover, a low CD62L/CD54 ratio correlated significantly with poor cytogenetics, p=0.006. In the group of MDS patients with <5% blasts, 4 developed a refractory anemia with excess of blasts or AML within a follow up period of 12 months. There was a trend for a lower CD62L/CD54 ratio for MDS patients who developed an AML compared with patients who did not. In conclusion, the CD62L/CD54 ratio is significantly decreased in MDS compared with healthy volunteers and even more decreased in AML with prior MDS. Both CD62L and CD54 are involved in regulation of proliferation and apoptosis of the HPC. A decreased adhesion ratio in low risk MDS patients might reflect HPC damage at an early stage of the disease with an increased proliferative capacity and a decreased apoptotic profile. Interestingly, a low CD62L/CD54 ratio showed a significant inverse correlation with the IPSS cytogenetic risk category. Due to an absence of metaphases in a proportion of MDS patients, cytogenetics is not always available. The CD62L/CD54 ratio might serve as a surrogate marker for poor prognosis cytogenetics in case no karyotype is available. Low risk MDS patients who developed an AML within 12 months tended to have a lower CD62L/CD54 ratio. Although these results are promising, sample size and follow up period needs to be extended. The CD62L/CD54 ratio might add to prognostication of MDS patients and might identify MDS patients with <5% blasts who are at risk for development of an AML. Disclosures: Ossenkoppele: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Van de Loosdrecht:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Bone Marrow Mesenchymal Stem Cell (BM-MSC) Release Microvesicles/Exosomes That Incorporate Into Hematopoietic Cells From MDS Patients and May Modify Their Behaviour

Blood ◽

10.1182/blood.v122.21.863.863 ◽

2013 ◽

Vol 122 (21) ◽

pp. 863-863 ◽

Cited By ~ 1

Author(s):

Sandra Muntión ◽

Post Doc Fellowship ◽

Teresa Ramos ◽

Bruno Paiva ◽

Beatriz Roson ◽

...

Keyword(s):

Bone Marrow ◽

Board Of Directors ◽

Research Funding ◽

De Novo ◽

Annexin V ◽

Micro Rna ◽

Bioactive Molecules ◽

Advisory Committees ◽

Micro Rnas ◽

Cd34 Cells

Abstract A new mechanism of intercellular communication has been proposed consisting in the secretion of exosomes/ microvesicles (MVs). Such mechanism has been shown to modify the functional properties of recipient cells by the transfer of proteins, mRNA, or micro-RNAs. The hypothesis of the present work was that MSC from MDS patients could differentially modify the HPC properties throughout the shedding of MVs when compared with those from controls due to their different content. Material and methods: MVs were isolated from MSC from bone marrow (BM) samples 18 patients diagnosed with ‘de novo’ and untreated low risk MDS and from MSC from 12 healthy BM. BM-MSC at third passage were cultured in DMEM deprived of FCS, and supernatants were collected after 6 or 24 hours. MVs purification was performed in the majority of the experiments (16 MDS/ 9 Controls) using the ExoQuick-TC exosome precipitation solution (ExoQuick; System Biosiences). To confirm the isolation of MVs by exosome precipitation solution, in some cases (2 MDS/3 Controls) the MVs were obtained by ultracentrifugation; MVs identification was done by transmission electron microscopy (TEM) as well as by flow cytometry (FC). To evaluate if the micro-RNA content into MSC-MVs from patients and controls was different, expression analysis of miRNAs was done using Megaplex™ RT Primers pool (Applied Biosystems) and 384-well microfluidic cards (TaqMan® MicroRNA Array A) were loaded with retro-transcription product and PCR runs were performed on a 7900HT Fast Real-time PCR system (eight MVs from MDS and 4 from HD).To demonstrate the incorporation of MVs from MSC into human hematopoietic progenitors (HPC: CD34+ cells obtained by immunomagnetic selection) HPC were co-cultured with MVs from MSC. Incorporation of Vybrant Dil-labelled MVs into HPC was evaluated at 1, 3, 6, and 24 h. by FC. To detect the incorporation of MVs by confocal microscopy (CM) an intracellular primary Ab for CD90 (Santa Cruz, Biotechnology) was used as MVs marker and anti-CD45 to detect HPC. A Zeiss LSM 510 CM connected to a digital camera (Leica DC 100) were used to obtain confocal images. Apoptotic rate of CD34+ cells that had the MVs-MSC from MDS and controls were evaluated by FC by using APC H7 Annexin V DY634 (Immunostep) and 7AAD (BD Biosciences). Results: More than 95% of MVs isolated by ExoKit system from supernatants of cultured MSC from 6 HD and 6 MDS patients showed scatter intensities lower than of 6µm beads. We observed, in all cases, the same FC pattern. Also, MVs/exosomes isolated by ultracentrifugation (3 MDS/ 5 HD) showed the same FC pattern. MVs from MDS and controls isolated by ultracentrifugation were identified by TEM (fig1). When co-cultures of CD34+ HPC and MVs were studied in both HD and MDS, MVs were incorporated into HPC in all cases (fig2). When the content of miRNAS in the MVs from MDS and HD were compared significant differences were observed between both groups. Twenty-one out of 384 evaluated miRNAs were over-expressed in the MVs from patients compared with the controls. To confirm these results, the expression of miR10a and miR-132 was analyzed by RT-PCR. In both cases their expression was significantly increased in MVs from patients. Recently, it has been suggested that the cargo of these structures are bioactive molecules, therefore we explored the possibility that MVs could modify the behavior of the target cell. For this purpose we searched in which pathways the overexpressed miRNAs could be involved and apoptosis was among them. Since it is considered a very important process in MDS pathophysiology we compared apoptosis by FC, after co-culturing CD34+ cells with MVs from MSC of MDS and HD. Interestingly, preliminary results show that the MVs from MDS protected better from apoptosis CD34+ cells than MVs obtained from controls. In summary, in the present study we show that BM-MSC produce MVs/exosomes with different microRNAs content according to their origin, MDS or HD. These structures can be incorporated into HPC and can modify their properties. Funding: Instituto de Salud Carlos III. PI12/01775. Junta de Castilla y León.GRS 873/A/13. Portuguese FST Grant. SFRH/BD/86451/2012 Disclosures: Diez-Campelo: Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. San Miguel:Jansen, Celgene, Onyx, Novartis, Millenium: Membership on an entity’s Board of Directors or advisory committees. del Cañizo:Celgene: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Jansen-Cilag: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Arry: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity’s Board of Directors or advisory committees, Research Funding.

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Extracellular Vesicles Play an Important Role in Intercellular Communication Between Bone Marrow Stroma and Hematopoietic Progenitor Cells in Myeloproliferative Neoplasms

Blood ◽

10.1182/blood.v128.22.1957.1957 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1957-1957

Author(s):

Teresa L. Ramos ◽

Luis Ignacio Sánchez-Abarca ◽

Beatriz Rosón ◽

Alba Redondo ◽

Concepción Rodríguez ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Board Of Directors ◽

Intercellular Communication ◽

Research Funding ◽

Hematopoietic Progenitor Cells ◽

Rt Pcr ◽

Hematopoietic Progenitor ◽

Advisory Committees ◽

Cd34 Cells

Abstract The complex interplay between bone marrow-derived mesenchymal stromal cells (BM-MSC) and neoplastic hematopoietic cells is involved in the progression of myeloproliferative neoplastic (MPN) diseases. Extracellular vesicles (EV) have emerged as a complex cell-to-cell communication system within the neoplastic microenvironment. EV are able to reprogram recipient cells by transferring proteins, mRNA and microRNA from their cell of origin. We aimed to analyze the microRNA content of EV obtained from MPN BM-MSC, as well as the changes induced when these EV are incorporated into hematopoietic progenitor cells (HPC). EV were isolated from BM-MSC of MPN patients (n=22) and healthy donors (HD) (n=19) by ultracentrifugation. Characterization of EV by transmission electron microscopy (TEM), immunoblot, multiparametric flow cytometry (MFC) and NanoSight analysis revealed vesicles with a typical bilayer-membrane characteristic morphology with a size inferior to 500 nm, which were positive for various EV markers as CD63 and CD81, and for MSC markers as CD73, CD90 and CD44 (Figure 1). MicroRNA profiling by 384-well microfluidic cards (TaqMan® MicroRNA Array A) showed an overall increase in the microRNA expression in the MPN-MSC-derived EV, when compared to EV from donor MSC. Using RT-PCR, we observed that miR-155 was selectively enriched in EV released by MPN-MSC. An overexpression of this microRNA was observed in EV (p=0.032), while a downregulation was observed in BM-MSC (p=0.0078) (Figure 2). EV incorporation was demonstrated by fluorescence microscopy and MFC, where HPC (CD34+ cells obtained by immunomagnetic selection) were co-cultured with Vybrant Dil-labeled EV. For functional studies apoptosis and clonogenic assays (CFU-GM) were performed. We observed an increase in CD34+ cell viability after incorporating EV from BM-MSC (HD and MPN). Moreover, an increase (p=0.04) in miR-155 expression was observed when HD HPC incorporated EV from MPN-MSC. When neoplastic CD34+ cells incorporated the EV derived from MPN-MSC an increase of CFU-GM number was also observed. We suggest that EV released from MPN-MSC represent a mechanism of intercellular communication between malignant stromal and hematopoietic cells, through the transfer of genetic information that may be relevant in the pathophysiology of these diseases. Funding: GRS 1034/A/14 (C. Sanidad, JCYL) and FCT (SFRH/BD/86451/2012) Figure 1 EV characterization by TEM (A), Immunobloting - CD63 (B) and MFC (C). Scale bar: 200 and 500 nm. Figure 1. EV characterization by TEM (A), Immunobloting - CD63 (B) and MFC (C). Scale bar: 200 and 500 nm. Figure 2 Expression of miR-155. RT-PCR from EV released from HD and MPN-MSC (A), and the expression of miR-155 in BM-MSC (B). Figure 2. Expression of miR-155. RT-PCR from EV released from HD and MPN-MSC (A), and the expression of miR-155 in BM-MSC (B). Disclosures Sánchez-Guijo: Bristol-Myers-Squib: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Incyte: Consultancy, Honoraria. Del Cañizo:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jansen-Cilag: Membership on an entity's Board of Directors or advisory committees, Research Funding; Arry: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Phase 2 Study of Monotherapy Galunisertib (LY2157299 Monohydrate) in Very Low-, Low-, and Intermediate-Risk Patients with Myelodysplastic Syndromes

Blood ◽

10.1182/blood.v126.23.1669.1669 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1669-1669 ◽

Cited By ~ 13

Author(s):

David Valcarcel ◽

Amit Verma ◽

Uwe Platzbecker ◽

Valeria Santini ◽

Aristoteles Giagounidis ◽

...

Keyword(s):

Bone Marrow ◽

Board Of Directors ◽

Research Funding ◽

Intermediate Risk ◽

Phase 2 ◽

Eligibility Criteria ◽

Advisory Committees ◽

Cd34 Cells ◽

Risk Patients ◽

Ecog Ps

Abstract Introduction: Myelodysplastic syndromes (MDS) are characterized by bone marrow dysplasia and ineffective hematopoiesis. Zhou et al. showed that transforming growth factor-beta (TGF-β) signaling was constitutively activated in MDS CD34+ cells and that this over-activation and subsequent myelosuppression was based on reduced expression of SMAD7, the natural inhibitor of TGF-β, in MDS CD34+ cells (Zhou L et al. Cancer Res 2011;71:955-963). Galunisertib specifically inhibited the kinase activity of the TGF-β receptor type I (TGF-βRI) also known as ALK5 and its downstream signaling pathway theoretically replaced the SMAD7 function. Galunisertib reversed hematopoietic suppression in human MDS bone marrow assays, and in a murine model of TGF-β derived bone marrow failure. Based on these preclinical studies that demonstrate hematological improvement (HI) in MDS models following galunisertib treatment, a single-arm phase 2 part of a phase 2/3 proof-of-concept study in very low-, low-, and intermediate-risk patients with MDS was conducted. Methods: The primary objective of this study was to estimate the HI rate based on International Working Group (IWG) 2006 criteria in patients with very low-, low-, and intermediate-risk MDS by Revised International Prognostic Scoring System (IPSS-R), treated with galunisertib. Eligible patients were treated with galunisertib 300 mg/day (150 mg BID) orally for 14 days, followed by 14 days off, constituting a cycle of 28 days. Eligibility criteria permitted any prior therapy, all of which were required to be discontinued at least 28 days prior to initiation of galunisertib. Supportive therapies including ongoing transfusions were allowed. Eligibility criteria included confirmed diagnosis of MDS, anemia with hemoglobin ≤10.0 g/dL, and an Eastern Cooperative Oncology Group performance status (ECOG PS) ≤2. Safety was assessed and summarized using the Common Terminology Criteria for Adverse Events (CTCAE v4.0). Descriptive statistics were used to report baseline characteristics and response rates. Results: In this phase 2 study, 41 patients received galunisertib orally (N=39, 150 mg BID and N=2, 80 mg BID for PK comparison). Patients were 62% males. The median age was 71 years (range: 52-84), the majority of patients were classified as refractory cytopenia with multilineage dysplasia (66.7%) or refractory anemia with ringed sideroblasts (20.5%) based on WHO MDS classification. ECOG PS was 0/1 in 53.8%/46.2% of patients. Sixty-two percent of the patients received ≥6 cycles of treatment. Among the 39 patients receiving 150 mg BID, a total of 15 (38%) patients discontinued from the study within 6 cycles; one due to AE and 9 due to patient/physician decision. The most common possibly related any grade treatment-emergent adverse events (TEAEs) included fatigue (20.5%), diarrhea (15.4%), pyrexia (10.3%), vomiting (10.3%), anemia (7.7%), nausea (7.7%), urinary tract infection (7.7%), neutrophil count decreased (5.1%), and platelet count decreased (5.1%); 12 (30.8%) patients had grade 3/4 TEAEs, 4 (10.3%) were drug-related. One of the 39 patients was protocol ineligible and was removed from the efficacy analysis. Among the 38 evaluable patients in the ITT population, 14 of whom required fewer than 4 units of transfusion per 8 weeks, 10/38 (26%) patients achieved HI, defined as at least a continuous 8-week response with at least a 4-unit reduction in transfusion requirement from baseline or hemoglobin increase by at least 1.5 g/dL per 8-week period. Of these 10 patients, 4 became transfusion-independent, and 5 had transfusion reduction. In a subgroup of 24/38 patients who had a transfusion requirement of at least 4 units every 8 weeks at baseline, 9 (38%) of these patients achieved a transfusion reduction of at least 4 units. No apparent correlation between cytogenetics or MDS subtype including ringed sideroblasts and response was identified; however, only 14 patients had abnormal cytogenetics. No platelet or neutrophil responses were observed. Conclusion: Galunisertib is well tolerated in this MDS population where this ALK5 inhibitor was investigated for the first time. Patients most commonly discontinued from study treatment due to patient/physician decision and not for toxicity. The clinical endpoint of HI was observed in 26% of the ITT population, and no specific response sub-group was identified. Disclosures Valcarcel: GSK: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; NOVARTIS: Honoraria, Membership on an entity's Board of Directors or advisory committees; AMGEN: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CELGENE: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Platzbecker:Boehringer: Research Funding; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Santini:celgene, Janssen, Novartis, Onconova: Honoraria, Research Funding. Díez-Campelo:Celgene: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau; Janssen: Research Funding. Schlenk:Boehringer-Ingelheim: Honoraria; Pfizer: Honoraria, Research Funding; Arog: Honoraria, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Teva: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees. Gaidano:MorphoSys; Roche; Novartis; GlaxoSmithKline; Amgen; Janssen; Karyopharm: Honoraria, Other: Advisory boards; Celgene: Research Funding. Perez de Oteyza:Eli Lilly and Company: Research Funding. Cleverly:Eli Lilly and Company: Employment, Equity Ownership. Chiang:Eli Lilly and Copany: Employment. Lahn:Eli Lilly and Company: Other: Former employee. Desiaih:Eli Lilly and Company: Employment. Guba:Eli Lilly and Company: Employment, Equity Ownership. List:Celgene Corporation: Honoraria, Research Funding. Komrokji:Pharmacylics: Speakers Bureau; Novartis: Research Funding, Speakers Bureau; Incyte: Consultancy; Celgene: Consultancy, Research Funding.

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PRM-151 in Myelofibrosis: Durable Efficacy and Safety at 72 Weeks

Blood ◽

10.1182/blood.v126.23.56.56 ◽

2015 ◽

Vol 126 (23) ◽

pp. 56-56 ◽

Cited By ~ 25

Author(s):

Srdan Verstovsek ◽

Ruben A. Mesa ◽

Lynda M Foltz ◽

Vikas Gupta ◽

John O. Mascarenhas ◽

...

Keyword(s):

Bone Marrow ◽

Adverse Events ◽

Board Of Directors ◽

Research Funding ◽

Negative Slope ◽

Computer Assisted ◽

Jak Inhibitor ◽

Efficacy And Safety ◽

Advisory Committees ◽

Grade 3

Abstract Background: PRM-151 (PRM) is a recombinant form of pentraxin-2, an endogenous human protein that acts at sites of tissue damage, inducing macrophage differentiation to prevent and reverse fibrosis. 27 patients with primary myelofibrosis (MF), post-essential thrombocythemia MF, or post-polycythemia vera MF and Grade 2 or 3 bone marrow (BM) fibrosis enrolled in the first stage of a 2-stage adaptive trial in which PRM-151 10 mg/kg IV was administered for 24 weeks in four different arms: PRM-151 QW (n=8), PRM-151 Q4W (n=7), PRM-151 QW + ruxolitinib (RUX) (n=6), or PRM-151 Q4W + RUX (n=6). At 24 weeks, reductions in BM fibrosis, improvements in hemoglobin (Hgb) and platelets (PLT), decreases in symptoms (MPN-SAF Total Symptom Score [TSS]), and modest reductions in spleen size by palpation were observed in all arms, with a favorable safety profile (Verstovsek, ASH 2014, Abstract 713). Patients experiencing clinical benefit were allowed to continue beyond 24 weeks. We now report efficacy and safety in 13 patients who have completed at least 72 weeks of treatment. Bone marrow fibrosis status by morphologic WHO grading and computer-assisted image analysis (CIA) are available up to 48 weeks in some patients, as assessed by central hematopathologist reviewers blinded to patient, treatment, and timepoint. BM data through 72 weeks is pending. WHO response was defined as ≥1 grade reduction in MF grade at any time and CIA response was defined as a decrease in the % fibrosis compared to baseline with a negative slope > 1. (Pozdnyakova, EHA 2015, Abstract P677). Baseline Demographics (N=13): Median age 60 (51-76); 46% DIPSS Int-1, 54% DIPSS Int-2; 62% PMF, 15% post-ET MF, 23% post-PV MF; 46% grade 3 BM fibrosis, Hgb < 100 g/L in 38%, PLT < 100 x 109/L in 69% and <50 x 109/L in 38%; 31% JAK inhibitor-naive and 69% received a prior or current JAK inhibitor. Study treatment (N=13): In the first 24 weeks, treatment was PRM-151 QW (n=5), PRM-151 Q4W (n=3), PRM-151 QW + RUX (n=2), PRM-151 Q4W + RUX (n=3). At 28 weeks, treatment changed to PRM-151 QW (n=2), PRM-151 Q4W (n=7), and PRM-151 Q4W + RUX (n=4), with 1 patient stopping RUX for thrombocytopenia. Both PRM-151 QW patients switched to PRM-151 Q4W after 40 and 52 weeks. Two patients missed weeks 64 and 68 due to complications of a motor vehicle accident and abdominal surgery, respectively, but are continuing treatment as of week 72. BM (n=13): 54% had a morphologic response, and 85% had a CIA response. Hgb (g/L): In 5 pts with baseline Hgb < 100, median Hgb increased by 24% from 86 (range 77-97) at baseline to 107 (range 71-113) at Week 72. 3 of 5 patients who were receiving transfusions at baseline became transfusion independent, with durations of 32-60 weeks. (Figure 1) PLT (x 109/L): In 9 pts with baseline < 100, median PLT count increased by 37% from 38 (range 10-89) at baseline to 52 (range 26-159) at Week 72. All 4 patients who were receiving PLT transfusions at baseline became transfusion independent, with durations of 24-44 weeks. (Figure 2) Symptoms (N=13): 69% and 38% of patients had ≥ 50% and 100% reductions from baseline in TSS between 24 and 72 weeks, with durations of up to 48 and 12 weeks, respectively. (Figure 3) Spleen (N= 9 with palpable spleens at baseline): 50% of pts had ≥25% reduction, 2 of whom had ≥50% reduction lasting > 12 weeks. (Figure 4) Safety (N=13): Most common adverse events (AEs) regardless of relatedness were fatigue (4), nausea (3), fever (3), cough (2), diarrhea (2), tooth infection (2), headache (2), upper respiratory infection (2), hyperglycemia (2), and hyperuricemia (2). There were 13 possibly or probably related adverse events in 3 patients from beginning of study through 71 weeks, 11 Grade 1, 1 Grade 2 and 1 Grade 3, with no event occurring in > 1 patient. There were no related serious AEs in these patients. Conclusion: In 13 patients completing at least 72 weeks, PRM-151 treatment was well tolerated, and improvements in Hgb, PLT, symptoms and spleen appeared to increase with longer treatment duration. Disclosures Mesa: Novartis Pharmaceuticals Corporation: Consultancy; NS Pharma: Research Funding; Gilead: Research Funding; Promedior: Research Funding; Genentech: Research Funding; CTI Biopharma: Research Funding; Incyte Corporation: Research Funding; Pfizer: Research Funding. Foltz:Promedior: Research Funding. Gupta:Incyte Corporation: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Promedior: Research Funding. Mascarenhas:Kalobios: Research Funding; Roche: Research Funding; Promedior: Research Funding; Novartis Pharmaceuticals Corporation: Research Funding; CTI Biopharma: Research Funding; Incyte Corporation: Research Funding. Ritchie:Incyte: Speakers Bureau; Celgene: Speakers Bureau. Hoffman:All Cells, LLC: Consultancy, Membership on an entity's Board of Directors or advisory committees; Geron: Consultancy, Membership on an entity's Board of Directors or advisory committees; Promedior: Research Funding. Silver:Promedior: Research Funding. Pozdnyakova:Promedior: Consultancy. Hasserjian:Promedior: Consultancy. Trehu:Promedior: Employment, Equity Ownership. Salama:Promedior: Consultancy. Gotlib:Allakos, Inc.: Consultancy.

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Enhanced and Accelerated Repopulation of Mutant C-Kit Immunodeficient Mice By Transplants of Primary Chronic and Acute Myeloid Leukemic Human Cells

Blood ◽

10.1182/blood.v128.22.4537.4537 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4537-4537 ◽

Cited By ~ 1

Author(s):

Naoto Nakamichi ◽

Colin A. Hammond ◽

Paul H. Miller ◽

Katharina Rothe ◽

Philip A Beer ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Hematopoietic Cells ◽

Leukemic Cells ◽

Advisory Committees ◽

Immunodeficient Mice ◽

Gm Csf ◽

Nsg Mice ◽

Cd34 Cells ◽

Normal Human

Abstract Background: Assessment of the growth and differentiation of human hematopoietic cells transplanted into immunodeficient mice has become an important method for evaluating the numbers and properties of cells with engraftment potential and the progeny they can generate in an in vivo setting. Such xenograft models have undergone numerous modifications and have also been applied with increasing success to primary sources of many types of malignant as well as normal human hematopoietic cells. These advances include the incorporation of cDNAs encoding various human-specific growth factor genes into the genomes of the widely used and long-lived NOD-scid-IL2Rγcnull (NSG) mouse, the creation of regenerated human bone ossicles within the mice to more closely mimic the human marrow microenvironment, recognition of the superior supportive activity of female mice, and more recently of NSG mice additionally compromised by their genetic acquisition of a c-kit deficiency. Exploitation of these modifications to recipients of human hematopoietic cells has greatly increased our understanding of the complex composition of both normal and leukemic populations and their responses to various treatments. Nevertheless, many examples remain of both acute and chronic human myeloid leukemias that do not engraft or engraft very poorly, even in the most permissive mice thus far tested. These include samples of many cases of chronic phase chronic myeloid leukemia (CP-CML), myelodysplastic syndromes (MDS), and approximately one third of all cases of acute myeloid leukemia (AML). We have now created 2 new strains of immunodeficient mice that have the B, T, NK immunodeficiency determined by a SirpαNOD-Rag1-/--IL2Rγc-/- genotype (which is equivalent to that of NSG mice) and a c-kit deficiency obtained by replacement of the wild-type c-kit gene with a homozygous W41/W41genotype (SRG-W41 mice). The second strain has the same SRG-W41 genotype but is also transgenic for human IL3, GM-CSF, and SCF production (SRG-W41-3GS). NRG/SRG mice were chosen as the parental strain because they support similar levels of chimerism as NSG mice when given a radiobiologically equivalent dose of radiation, but can benefit from split or low dose rate irradiation protocols. Results: We first showed that leukemic cells from patients with AML and CP-CML produce progeny more rapidly in sub-lethally irradiated parental SRG-3GS mice than in SRG mice, and that SRG-W41 mice can support higher levels of human myeloid and B-lymphoid chimerism by normal human CD34+ cord blood (CB) and adult bone marrow (BM) cells than similarly suppressed or non-irradiated SRG controls. We then compared the chimerism obtained in different strains of mice transplanted each with 6x106 CD34+ cells from 1 CP-CML patient or ~106 CD34+ cells from 5 different AML patients pre-selected for their previously demonstrated poor or inability to engraft NSG-3GS mice. Within a few weeks, the level of the predominantly myeloid chimerism in 2 non-irradiated male SRG-W41-3GS mice and 2 sub-lethally irradiated female SRG-3GS recipients became equivalent and this equivalence was sustained for over a year. Only 3 of 5 AML samples tested engrafted either sex of sub-lethally irradiated SRG-W41-3GS or SRG-3GS mice, and in each case, the speed and level of engraftment was greatly enhanced in the SRG-W41-3GS mice as compared to sex-matched SRG-3GS hosts (with 20-30-fold and 2-3-fold higher levels at week 6 and 10 respectively in female as compared to male recipients of either strain). All the engrafted SRG-W41-3GS female hosts became moribund within 20 weeks post-transplant with a predominance of human leukemic cells in their BM and enlarged spleens filled with leukemic cells. In contrast, the SRG-3GS recipients of the same AML cells survived more than 20 weeks with a more delayed appearance of leukemic cells. Sublethally irradiated female SRG-W41-3GS recipients of 0.1-0.2x106 CD34+ cells from 3 of 3 chronic myelomonocytic leukemia (CMML) patients tested to date have also shown engraftment at either week 3 or 11 and, in one case, up to 17 weeks post-transplant. Conclusion: The introduction of a c-kit deficiency into highly immunodeficient SRG mice producing human IL3, GM-CSF and SCF transgenically offers new opportunities for examining the growth potential and sensitivity to treatment of a broader spectrum of primary human leukemias and neoplastic conditions than has been previously possible. Disclosures Deininger: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; CTI BioPharma Corp.: Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding; BMS: Consultancy, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees.

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Increased Interleukin-8 (IL8)-CXCR2 Signaling Promotes Progression of Bone Marrow Fibrosis in Myeloproliferative Neoplasms

Blood ◽

10.1182/blood-2020-138843 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 6-7

Author(s):

Andrew Dunbar ◽

Min Lu ◽

Mirko Farina ◽

Young Park ◽

Julie Yang ◽

...

Keyword(s):

Bone Marrow ◽

Overall Survival ◽

Board Of Directors ◽

Research Funding ◽

Myeloproliferative Neoplasms ◽

Private Company ◽

Advisory Committees ◽

Cd34 Cells

Introduction: Elevated pro-inflammatory cytokines are a hallmark feature of myeloproliferative neoplasms (MPNs). The pro-inflammatory cytokine interleukin-8 (IL8) is increased in patients with myelofibrosis (MF) and correlates with adverse outcome, including overall survival. Previously, the Levine/Fang labs identified increased IL8 secretion from individual CD34+ stem cells in a subset of MF patients. The role of IL8 and its cognate receptors CXCR1/2 in MF pathogenesis has not been delineated. Methods: Single-cell cytokine assays were performed on isolated CD34+ cells from 60 clinically annotated MPN patients (20 MF, 20 PV, 20 ET) using a previously described micro-chip platform (Kleppe et al, Can Disc 2013). 10 healthy donors (CD34+ cells from hip replacements) were used as controls. Integrated RNA-Seq and Assay for Transposase-Accessible Chromatin followed by next-generation sequencing (ATAC-Seq) was performed on CD34+ cells from MPN patients with and without expanded IL8 secreting clones for gene expression/chromatin accessibility analysis. To model the role of IL8-CXCR2 on fibrosis in vivo, the human MPLW515L transplant model (hMPLW515L) of MF was used. Specifically, wild-type (WT) murine bone marrow (Creneg-Cxcr2f/f; Cxcr2WT) or marrow lacking the CXCR2 receptor (VavCre-Cxcr2f/f; Cxcr2KO)were retrovirally infected with MSCV-hMPLW515L-IRES-GFP and transplanted into lethally irradiated WT recipient mice and monitored for disease. Blood counts, chimerism, and flow cytometry were assayed. Moribund mice were sacrificed and assayed for grade reticulin fibrosis and overall survival. Results: Single-cell cytokine assays confirmed an increased proportion of IL8-secreting CD34+ cells in MF patients (40%) in comparison to other MPN sub-types (10% PV/0% ET) (Figure 1A). MF patients with expanded IL8 secreting clones (defined as >50% of total CD34+ cells) had increased leukocytosis (p<0.0001), larger spleen sizes (p=0.0004), greater prevalence of constitutional symptoms (p=0.0084), and higher-grade reticulin fibrosis in marrow (Figure 1B) in comparison to MF patients without prevalent IL8 clones. IHC confirmed increased IL8 expression in marrow biopsies from 8/15 MF patients in comparison to 0/4 normal controls (Figure 1C), and high IL8 expression was also observed in MF splenic megakaryocytes (MKs) as well as in splenic stromal/endothelial cells not seen in normal spleen (Figure 1D). Integrated RNA-Seq/ATAC-Seq analysis of IL8-high MF patients confirmed up-regulation of IL8-CXCR2 signaling and enrichment in pro-inflammatory pathways (i.e TNFa, NFkB, etc) by GSEA, as well as increased expression/accessibility of pro-inflammatory genes S100A8 and S100A9-previously implicated in fibrosis development. Flow analysis of IL8-high MF CD34+ cells revealed enhanced surface expression of CXCR2 and its analog CXCR1, such that MF was characterized by increased IL8 ligand and receptor expression (Figure 1E) and coincided with enhanced NFkB pathway activity (Figure 1F). Consistent with this, colony forming assays of cultured MF CD34+ cells revealed enhanced colony output when cultured with IL8 compared to WT CD34+ cells-an effect ameliorated by co-treatment with the CXCR1/2 antagonist Reparixin (Figure 1G). In vivo, hMPLW515L adoptive transplant with Cxcr2KO hematopoietic donor cells demonstrated improved leukocytosis, thrombocytosis (Figure 2A) and splenomegaly in comparison to Cxcr2WT hMPLW515L recipient mice. Pathologic analysis revealed a reduction in reticulin fibrosis in bone marrow (Figure 2B) and spleen, translating into an improvement in overall survival (Figure 2C). Notably, a significant reduction in dysplastic MKs-a hallmark feature of MF-was also observed in Cxcr2KO hMPLW515L mice (Figure 2D) supporting a role for CXCR2 signaling in MK proliferation. Conclusion: IL8 secreting clones are associated with increased symptom severity and fibrosis grade in MF. Gene expression of MF CD34+ IL8 secreting clones shows up-regulation of inflammatory genes S100A8/A9, implicated in myofibroblast proliferation. Cxcr2 KO abrogates fibrosis formation and prolongs survival in the hMPLW515L model, and CXCR1/2 inhibition impairs colony forming capacity of MF CD34+ cells. These data suggest pharmacologic inhibition of this pathway should be investigated as potential therapy in MF and in PV/ET patients at high risk of fibrotic transformation. Disclosures Fan: IsoPlexis: Current Employment, Current equity holder in private company; Singleron Biotechnologies: Current Employment, Current equity holder in private company. Levine:Morphosys: Consultancy; Prelude Therapeutics: Research Funding; Qiagen: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria; Loxo: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Amgen: Honoraria; Astellas: Consultancy; Imago: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Isoplexis: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Lilly: Consultancy, Honoraria; Janssen: Consultancy. Hoffman:Protagonist: Consultancy; Abbvie: Membership on an entity's Board of Directors or advisory committees; Dompe: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Forbius: Consultancy.

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