Immune Thrombocytopenia (ITP) and Hβ-1 Tubulin: The Arg307His Substiution Is Over-Represented and Identifies Patients with More Severe Disease

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2525-2525
Author(s):  
Paul Andrew Basciano ◽  
Luis Javier Leandro-Garcia ◽  
Cristina Rodriguez-Antona ◽  
Paraskevi Giannakakou ◽  
James B. Bussel
Keyword(s):  
Board Of Directors ◽  
Research Funding ◽  
Severe Disease ◽  
Wild Type ◽  
Advisory Committees ◽  
Platelet Production ◽  
Equity Ownership ◽  
And Function ◽  
Β Tubulin

Abstract Abstract 2525 Platelet production and function are dependent on the presence of the hematopoietic-specific β-tubulin isotype Hβ1 (Class VI), whose expression is restricted to platelets and megakaryocytes, constituting 90% of total platelet β-tubulin. Among the eight human β-tubulin isotypes, Hβ1 is the only isotype for which frequent non-synonymous single nucleotide polymorphisms (SNPs) have been described. Little is known regarding the role these SNPs play in platelet production and function. In ITP, It is accepted that both accelerated platelet destruction and decreased platelet production are central to the disease process but the exact pathophysiologic mechanisms underlying individual patient variation are unknown. Likewise, little is understood regarding the diverse clinical manifestations of ITP—which range from minor to life-threatening bleeding, and even thrombosis—among patients who otherwise appear to have similar disease. The central role of platelets in ITP together with the platelet-specific expression of Hβ1 tubulin prompted us to investigate the potential role of Hβ1 SNPs in the pathophysiology and disease manifestations of ITP. We sequenced the coding region of Hβ1 gene using genomic DNA extracted from whole blood of 98 mostly-Caucasian ITP patients and 360 Caucasian controls. Our results showed that one of the 6 reported SNPs, namely 27795494G>A leading to the substitution of arginine for histidine at amino acid 307 (Arg307His), was overrepresented in the ITP patient population as compared to the controls. Specifically, the A allele was overrepresented in the ITP population (A allele frequency of 19% versus 16%; p=0.04). Importantly, the frequency of the homozygous A/A genotype was also significantly higher in the ITP population compared to the control population (7.1% versus 3.9%; p=0.05), while we did not find any changes in the frequencies of the heterozygote and wild-type genotypes. We retrospectively examined the disease characteristics of the different genotype populations in the ITP group; namely the homozygous (A/A) Hβ1 SNP versus the heterozygote (A/G) and homozygote wild-type (G/G) groups, which were combined after all analyses showed them to be similar, and are herein referred to as A/G+G/G. Our analysis showed no significant differences in gender, age, age at initial ITP diagnosis and time since initial diagnosis, between the A/A and A/G+G/G genotypes; there was also no difference in the incidence of concurrent autoimmune conditions between the groups. However, we found that the percentage of patients with platelet counts less than 30K/μL at disease presentation was significantly higher in the A/A genotype compared to the combined A/G+G/G genotype group (100% versus 55%, p=0.035). Furthermore, the total number of different treatment types for ITP within each group was significantly different, with A/A patients requiring a mean of 7.6 treatments (range 4–14), while the A/G+G/G patients required a mean of 5.4 treatments (range, 0–14) (p=0.03) over the course of their diseases. There were no significant differences between the groups with regard to responses to individual treatments (intravenous immune globulin, anti-D, rituximab, eltrombopag, and romiplostim). Taken together, these results suggest that alterations in Hβ1 tubulin play a patholphysiologic role in the development of ITP and that patients with the homozygous A/A genotype have more severe disease at presentation and more difficulty with maintenance of long-term disease control. Disclosures: Bussel: Amgen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; GlaxoSmithKline: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ligand: Membership on an entity's Board of Directors or advisory committees, Research Funding; Shionogi: Membership on an entity's Board of Directors or advisory committees, Research Funding; Eisai: Membership on an entity's Board of Directors or advisory committees, Research Funding; Cangene: Research Funding; Genzyme: Research Funding; IgG of America: Research Funding; Immunomedics: Research Funding; Sysmex: Research Funding.

scholarly journals Nfe2 Is Dispensable for Early, but Required for Adult Thrombocyte Formation and Function in Zebrafish

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2534-2534
Author(s):  
Megan S Rost ◽  
Ilya Shestopalov ◽  
Yang Liu ◽  
Andy H Vo ◽  
Francesca Barrett ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Endothelial Injury ◽  
Wild Type ◽  
Advisory Committees ◽  
High Fecundity ◽  
Platelet Production ◽  
Zebrafish Larvae ◽  
Equity Ownership ◽  
And Function

Abstract The NFE2 transcription factor is expressed in multiple hematopoietic lineages with a well-defined role in regulating megakaryocyte biogenesis and platelet production in mammals. Mice deficient in NFE2 completely lack circulating platelets, causing early lethality due to neonatal hemorrhage. Recent data in mice suggest some differences in embryonic and adult thrombopoiesis, and overexpression of NFE2 in murine bone marrow cells increases megakaryocyte maturation and platelet release, suggesting a role for NFE2 in both early and late megakaryocyte development. Zebrafish have emerged as an excellent model for studying hematopoiesis and thrombopoiesis due to their external development, optical transparency, high fecundity, and conservation of nearly the entire hemostatic system. Rather than platelets, zebrafish possess thrombocytes - nucleated cells believed to be the functional equivalent in mammals. We designed TALENs to target exon 4 of zebrafish nfe2, producing two mutant strains containing either an 8 or 10 base pair deletion, both resulting in a frameshift and null allele. We tracked survival for over one year and found that unlike mammals, zebrafish survive into adulthood in the absence of Nfe2 function with no signs of overt bleeding or lethality. We bred the nfe2 mutation into a transgenic background in which thrombocytes and hematopoietic progenitor cells express green fluorescent protein (Tg(cd41:GFP)) and are characterized by GFPhigh and GFPlow expression, respectively. We performed flow cytometry analysis and found that the percentage of GFPhigh cells (circulating thrombocytes) in the peripheral blood was significantly decreased from 0.67% to 0.2% in homozygous mutants (p < 0.001). In contrast, the percentage of GFPlow cells in the kidney marrow, the site of hematopoiesis in adult zebrafish, was increased from 0.47% to 1.17% in nfe2-/- mutants (p < 0.001). Surprisingly, quantification of circulating thrombocytes in 6 day old nfe2 null zebrafish larvae showed no significant differences from wild type siblings. Finally, we performed colony forming assays on whole kidney marrow lysates to measure the ability of hematopoietic progenitors to differentiate into thrombocytes. Both mutant and wild type adults are capable of producing thrombocytic colonies in the presence of thrombopoietin and erythropoietin. We and others have shown that thrombocytes participate in the formation of induced thrombi upon laser-mediated endothelial injury in zebrafish embryos and larvae. We tested the functionality of nfe2-/- thrombocytes and were surprised to find that wild type and nfe2 null zebrafish larvae form fibrin- and thrombocyte-rich clots in response to endothelial injury at day of life 3 (venous circulation) and 6 (arterial circulation), respectively. Measurement of both the time to occlusion as well as the total number of thrombocytes adhering to the site of injury revealed no significant differences between wild type and nfe2-/- larvae. These data suggest that loss of Nfe2 results in a late block in thrombopoiesis with secondary expansion of thrombocytic precursors, both features that are consistent with mammals. Surprisingly, Nfe2 appears to be dispensable for early embryonic thrombocyte production and function. These results suggest parallels with erythropoiesis, including distinct primitive and definitive pathways of development. This includes the potential for a previously unknown Nfe2-independent pathway of embryonic thrombopoiesis. The long term homozygous mutant survival will also facilitate more in depth study of Nfe2 deficiency in vivo, and further investigation could lead to alternative methodologies for the enhancement of platelet production in vivo or ex vivo. Disclosures Zon: Fate, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Scholar Rock: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Marauder Therapeutics: Equity Ownership, Other: Founder.

scholarly journals Role of Remission Status and Prior Transplant in Optimizing Survival Outcomes Following Allogeneic Hematopoietic Stem Transplantation (HSCT) in Patients Who Received Inotuzumab Ozogamicin (INO) for Relapsed / Refractory (R/R) Acute Lymphoblastic Leukemia (ALL)

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 886-886
Author(s):  
Partow Kebriaei ◽  
Matthias Stelljes ◽  
Daniel J. DeAngelo ◽  
Nicola Goekbuget ◽  
Hagop M. Kantarjian ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Survival Probability ◽  
Research Funding ◽  
Employment Equity ◽  
Inotuzumab Ozogamicin ◽  
Advisory Committees ◽  
Allogeneic Hsct ◽  
Post Transplant ◽  
Equity Ownership

Abstract Introduction: Attaining complete remission (CR) prior to HSCT is associated with better outcomes post-HSCT. Inotuzumab ozogamicin (INO), an anti-CD22 antibody conjugated to calicheamicin, has shown significantly higher remission rates (CR/CRi and MRD negativity) compared with standard chemotherapy (SC) in patients (pts) with R/R ALL (Kantarjian et al. N Engl J Med. 2016). Pts treated with INO were more likely to proceed to HSCT than SC, which allowed for a higher 2-yr probability of overall survival (OS) than patients receiving SC (39% vs 29%). We investigated the role of prior transplant and proceeding directly to HSCT after attaining remission from INO administration as potential factors in determining post-HSCT survival to inform when best to use INO in R/R ALL patients. Methods: The analysis population consisted of R/R ALL pts who were enrolled and treated with INO and proceeded to allogeneic HSCT as part of two clinical trials: Study 1010 is a Phase 1/2 trial (NCT01363297), while Study 1022 is the pivotal randomized Phase 3 (NCT01564784) trial. Full details of methods for both studies have been previously published (DeAngelo et al. Blood Adv. 2017). All reference to OS pertains to post-HSCT survival defined as time from HSCT to death from any cause. Results: As of March 2016, out of 236 pts administered INO in the two studies (Study 1010, n=72; Study 1022, n=164), 101 (43%) proceeded to allogeneic HSCT and were included in this analysis. Median age was 37 y (range 20-71) with 55% males. The majority of pts received INO as first salvage treatment (62%) and 85% had no prior SCT. Most pts received matched HSCTs (related = 25%; unrelated = 45%) with peripheral blood as the predominant cell source (62%). The conditioning regimens were mainly myeloablative regimens (60%) and predominantly TBI-based (62%). Dual alkylators were used in 13% of pts, while thiotepa was used in 8%. The Figure shows post-transplant survival in the different INO populations: The median OS post-HSCT for all pts (n=101) who received INO and proceeded to HSCT was 9.2 mos with a 2-yr survival probability of 41% (95% confidence interval [CI] 31-51%). In patients with first HSCT (n=86) the median OS post-HSCT was 11.8 mos with a 2-yr survival probability of 46% (95% CI 35-56%). Of note, some patients lost CR while waiting for HSCT and had to receive additional treatments before proceeding to HSCT (n=28). Those pts who went directly to first HSCT after attaining remission with no intervening additional treatment (n=73) fared best, with median OS post-HSCT not reached with a 2-yr survival probability of 51% (95% CI 39-62%). In the latter group, 59/73 (80%) attained MRD negativity, and 49/73 (67%) were in first salvage therapy. Of note, the post-HSCT 100-day survival probability was similar among the 3 groups, as shown in the Table. Multivariate analyses using Cox regression modelling confirmed that MRD negativity during INO treatment and no prior HSCT were associated with lower risk of mortality post-HSCT. Other prognostic factors associated with worse OS included older age, higher baseline LDH, higher last bilirubin measurement prior to HSCT, and use of thiotepa. Veno-occlusive disease post-transplant was noted in 19 of the 101 pts who received INO. Conclusion: Administration of INO in R/R ALL pts followed with allogeneic HSCT provided the best long-term survival benefit among those who went directly to HSCT after attaining remission and had no prior HSCT. Disclosures DeAngelo: Glycomimetics: Research Funding; Incyte: Consultancy, Honoraria; Blueprint Medicines: Honoraria, Research Funding; Takeda Pharmaceuticals U.S.A., Inc.: Honoraria; Shire: Honoraria; Pfizer Inc.: Consultancy, Honoraria, Research Funding; Novartis Pharmaceuticals Corporation: Consultancy, Honoraria, Research Funding; BMS: Consultancy; ARIAD: Consultancy, Research Funding; Immunogen: Honoraria, Research Funding; Celgene: Research Funding; Amgen: Consultancy, Research Funding. Kantarjian: Novartis: Research Funding; Amgen: Research Funding; Delta-Fly Pharma: Research Funding; Bristol-Meyers Squibb: Research Funding; Pfizer: Research Funding; ARIAD: Research Funding. Advani: Takeda/ Millenium: Research Funding; Pfizer: Consultancy. Merchant: Pfizer: Consultancy, Research Funding. Stock: Amgen: Consultancy; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Wang: Pfizer: Employment, Equity Ownership. Zhang: Pfizer: Employment, Equity Ownership. Loberiza: Pfizer: Employment, Equity Ownership. Vandendries: Pfizer: Employment, Equity Ownership. Marks: Pfizer: Consultancy, Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria, Speakers Bureau.

Final Results from an International, Multi-Center, Single-Arm Study Evaluating the Safety and Efficacy of Romiplostim in Adults with Primary Immune Thrombocytopenia (ITP),

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3279-3279 ◽  
Author(s):  
Ann Janssens ◽  
Michael D. Tarantino ◽  
Robert Bird ◽  
Maria Gabriella Mazzucconi ◽  
Ralph Vincent V. Boccia ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Platelet Count ◽  
Board Of Directors ◽  
Research Funding ◽  
Platelet Response ◽  
Employment Equity ◽  
Advisory Committees ◽  
Platelet Production ◽  
Platelet Counts ◽  
Equity Ownership

Abstract Abstract 3279 Background: ITP is an autoimmune disorder characterized by increased platelet destruction and suboptimal platelet production. Romiplostim stimulates platelet production via the TPO-receptor, and is recommended for second- and third-line treatment of chronic ITP in adults. We report final data from a large prospective study of romiplostim in adults with ITP of varying duration and severity. Methods: Eligibility criteria were broad: patients ≥18 years of age, who had received prior ITP therapies (final protocol amendment: ≥1, previous amendments: ≥3), with low platelet counts (final amendment: ≤ 30 × 109/L, previous amendments: ≤ 10, ≤ 20 × 109/L) or experiencing uncontrolled bleeding. The only excluded comorbidities were: hematological malignancy, myeloproliferative neoplasms, MDS and bone marrow stem cell disorder. Romiplostim was initiated at 1 (final amendment) or 3 (previous amendments) μg/kg/week, with dose adjustments allowed to maintain platelet counts ≥50 × 109/L. Patients could continue on study until they had access to commercially available romiplostim. Rescue medications were allowed at any time; concurrent ITP therapies could be reduced when platelet counts were > 50 × 109/L. Primary endpoint was incidence of adverse events (AEs) and antibody formation. Secondary endpoint was platelet response, defined as either (1) doubling of baseline count and ≥ 50 × 109/L or (2) ≥20 × 109/L increase from baseline. Results: A total of 407 patients received romiplostim, 60% of whom were female. Median (Q1, Q3) time since ITP diagnosis was 4.25 (1.20, 11.40) years (maximum 57.1 years), with 51% of patients splenectomised and 39% receiving baseline concurrent ITP therapies. Seventy-one percent of patients completed the study, with requirement for alternative therapy and withdrawn consent the most common reasons for discontinuation (5% each). Median (Q1, Q3) on-study treatment duration was 44.29 (20.43, 65.86) weeks (maximum 201 weeks), with a total of 20,201 subject-weeks on study. Incidence and type of AEs were consistent with previous studies. The most common serious treatment-related AEs were cerebrovascular accident, headache, bone marrow reticulin fibrosis (with no evidence of positive trichrome staining for collagen and no evidence suggesting primary idiopathic myelofibrosis), nausea, deep vein thrombosis, hemorrhage and pulmonary embolism, with each reported in 2 of 407 (0.5%) patients. All other serious treatment-related AEs were each reported in one patient. Eighteen patients died; 3 deaths (hemolysis, intestinal ischaema, aplastic anemia) were considered treatment-related. No neutralizing antibodies to romiplostim or TPO were reported. Approximately 90% of patients achieved each of the platelet response definitions, regardless of splenectomy status. Overall, median (Q1, Q3) time to response was 2 (1, 4) weeks for response definition 1, and 1 (1, 3) week for response definition 2. Median (Q1, Q3) baseline platelet count was 14 (8, 21) × 109/L. After 1 week of treatment median (Q1, Q3) platelet count had increased to 42 (18, 101) × 109/L. From week 8 onwards, and excluding counts within 8 weeks of rescue medication use, median platelet counts were consistently above 100 × 109/L (range 101.0–269.5 × 109/L). Median (Q1, Q3) average weekly romiplostim dose was 3.62 (1.99, 6.08) μg/kg. Summary/conclusions: This is the largest prospective study in adult ITP reported to date. The data reported here are similar to those reported for previous romiplostim studies, with romiplostim able to safely induce a rapid platelet response in adult ITP patients with low platelet counts or bleeding symptoms. Romiplostim is an important, well-tolerated, treatment option for adult ITP patients, which significantly increases and maintains platelet counts. Adverse Event Subject Incidence Platelet Response Disclosures: Janssens: Amgen: Consultancy; Roche: Speakers Bureau; GSK: Membership on an entity's Board of Directors or advisory committees. Tarantino:Cangene corporation: Research Funding; Baxter: Research Funding; Talecris: Honoraria, Speakers Bureau; Up-to-date: Patents & Royalties; The Bleeding and Clotting Disorders Institute: Board Member. Bird:Amgen: Honoraria, 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, Speakers Bureau; GSK: Membership on an entity's Board of Directors or advisory committees. Boccia:Amgen: Equity Ownership, Honoraria, Speakers Bureau. Lopez-Fernandez:Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Kozak:Amgen: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Steurer:Amgen: Honoraria. Dillingham:Amgen Limited: Employment, Equity Ownership. Lizambri:Amgen: Employment, Equity Ownership.

Absence Of Mutation In Cereblon (CRBN) and DNA Damage Binding Protein 1 (DDB1) Genes In Myeloma Cells and Patients and Its Clinical Significance

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3139-3139
Author(s):  
Anjan Thakurta ◽  
Anita K Gandhi ◽  
Michelle Waldman ◽  
Chad C. Bjorklund ◽  
Suzanne Lentzsch ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Heterozygous Mutation ◽  
Employment Equity ◽  
Advisory Committees ◽  
Single Nucleotide ◽  
Truncating Mutation ◽  
Equity Ownership

Abstract Background CRBN, a target of thalidomide and IMiDs® immunomodulatory agents lenalidomide (LEN) and pomalidomide (POM), is a component of the E3 ubiquitin cullin 4 ring ligase (CRL4) complex that also includes DDB1, Roc1, and Cul4. Two CRBN mutations have been reported in multiple myeloma (MM) patients: truncating mutation (Q99) and point mutation (R283K). One copy of the CRBN gene was shown to be deleted in the MM1S and MM1S.R cell lines. No DDB1 mutation has been described previously. Results We investigated the incidence of CRBN and DDB1 mutations by next-generation sequencing in 20 MM cell lines and MM subjects. Of 90 MM patients, 24 were newly diagnosed and 66 were relapsed and refractory of which 36 patients were LEN resistant. Out of the cell lines tested, 1 heterozygous CRBN mutation (D249Y) was found in the LEN-resistant ANBL6R cells, which is located in the putative DDB1 binding domain, and 2 single silent mutations were identified in the KMS-12-BM (rs17027638) and OPM-2 cells. One DDB1 heterozygous mutation (E303D) was identified in ANBL6 cells. In the cohort of patients assessed, no CRBN mutation was detected; however, 5 single nucleotide variations (SNV) were identified. Three of the 5 SNVs were at position 735 (Y245Y) and 1 each at position 219 (H73H) and 939 (C313C), respectively. The first 2 SNVs (rs17027638 and rs1045309) are described but not the last. We found a single SNV (P51P; rs2230356) in DDB1 gene the patient samples. Conclusion Mutations within the coding sequences of CRBN and DDB1 are rare in MM patients and cell lines. Most intrinsically LEN-resistant cells and cell lines made resistant to LEN or POM do not have CRBN or DDB1 mutations, suggesting the potential role of other sources, such as genetic or epigenetic pathways in developing resistance to IMiD drug–based therapy. Disclosures: Thakurta: Celgene: Employment, Equity Ownership. Gandhi:Celgene: Employment, Equity Ownership. Waldman:Celgene: Employment, Equity Ownership. Bjorklund:Celgene: Employment, Equity Ownership. Lentzsch:Celgene: Research Funding. Schey:Celgene: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; NAPP: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; BMS: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau. Orlowski:Bristol-Myers Squibb: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Millennium: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Onyx: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Resverlogix: Research Funding; Array: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Merck: Membership on an entity’s Board of Directors or advisory committees. Madan:Covance Genomics Lab: Employment. Ning:Celgene: Employment, Equity Ownership. Mendy:Celgene: Employment, Equity Ownership. Lopez-Girona:Celgene: Employment, Equity Ownership. Schafer:Celgene: Employment, Equity Ownership. Avet-Loiseau:Celgene: Research Funding. Chopra:Celgene: Employment, Equity Ownership.

Clot-On-A-Chip: A Microfluidic Device To Study Platelet Aggregation and Contractility Under Shear

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2363-2363 ◽  
Author(s):  
Lucas Ting ◽  
Shirin Feghhi ◽  
Ari Karchin ◽  
Wes Tooley ◽  
Nathan J White ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Board Of Directors ◽  
Microfluidic Device ◽  
Platelet Function ◽  
Research Funding ◽  
Force Generation ◽  
Advisory Committees ◽  
Equity Ownership ◽  
Contractile Forces

Abstract Introduction In primary hemostasis, platelets adhere, activate, and aggregate at the wall of an injured vessel to form a hemostatic plug for the cessation of bleeding. After activation, platelets generate myosin-driven contractile forces to compact the size of the plug in order to reduce the space between platelets and prevent their disaggregation. Hemodynamic shear can be a major effector of platelet function in hemostasis, but its effect on the ability of platelets to produce contractile forces is an open question. Studying the dynamics of platelet aggregation and platelet force generation under hemodynamic shear can provide important insights into hemostasis and thrombosis. Method We have developed a microfluidic device that uses microscale blocks to induce platelet aggregation and microscale posts to measure platelet forces in a hemostatic plug. Whole human blood in heparin or citrate is pumped through a microfabricated chip containing microchannels with arrays of blocks and posts arranged along the bottom of a microchannel (Fig. 1). The surface of the blocks and posts are pre-coated with von Willebrand factor and type I collagen to allow for platelet adhesion. As blood is passes over a block, its rectangular shape induces a high shear rate that causes platelets to aggregate on its surface. A flexible micropost is situated behind each block. As platelets aggregate between the block and post, their contractile forces causes the post to bend toward the block. The deflection of the post is recorded under fluorescence microscopy and analyzed using quantitative image analysis of the videos. Since a microscale post bends like a cantilever beam, its deflection can be used to quantify the forces of platelets. Results Blebbistatin, a myosin inhibitor, was used to confirm that deflection of the posts by the platelets in heparinized blood was due to myosin activity. When blood was incubated with 2-MeSAMP, a P2Y12 antagonist, platelets were able to aggregate, but their ability to generate contractile forces was substantially reduced. This finding indicates that ADP activation is needed for platelet contractility under shear. The rate of hemodynamic shear was found to influence platelet function, for the rate of platelet aggregation and force generation were found to increase for blood sheared from 2000 to 12,000 s-1. Moreover, platelet aggregation and contractile forces were reduced when glycoprotein Ib-V-IX complex and integrin αIIbβ3 were inhibited with antibody AK2 and antibody fragment c7E3 Fab, respectively. When citrated blood was incubated with tissue plasminogen activator, platelets aggregate and produced contractile forces that increased steadily within the first ten minutes, but then the forces began to subside. Conclusions Our device can be used to study the role of hemodynamic shear in platelet function and gives insights into the role of platelet forces during hemostasis. Its microscale dimensions also allow us the study the biomechanics involved in the formation of a hemostatic plug during its early stages of growth and stability. Disclosures: White: Vidacare Corp: Honoraria; Stasys Medical Corp: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties; NIH: Research Funding; Coulter Foundation: Research Funding; Washington State Life Sciences Discovery Fund: Research Funding. Sniadecki:Stasys Medical Corporation: Equity Ownership, Founder Other, Membership on an entity’s Board of Directors or advisory committees.

scholarly journals Myeloid-Lineage Enhancers Drive Oncogene Synergy and Represent a Novel Therapeutic Target in CSF3R-CEBPA Mutant AML

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 543-543
Author(s):  
Theodore Braun ◽  
Cody Coblentz ◽  
Sarah A Carratt ◽  
Mariam Okhovat ◽  
Amy Foley ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Board Of Directors ◽  
Median Survival ◽  
Stock Options ◽  
Research Funding ◽  
Wild Type ◽  
Advisory Committees ◽  
Dana Farber Cancer Institute ◽  
Equity Ownership ◽  
Cebpa Mutations

Acute Myeloid Leukemia (AML) results from the stepwise accumulation of mutations from distinct functional classes, ultimately culminating in malignant transformation. Based on their oncogenic activity, mutations can be classified into three distinct groups. Class I mutations activate signaling pathways, produce uncontrolled proliferation, and in isolation produce a myeloproliferative phenotype. Class II mutations result from point mutations or chromosomal translocation events in lineage determining transcription factors, producing differentiation arrest and myelodysplasia in isolation. A classic example of oncogene synergy between distinct mutational classes can be found in the co-occurrence of mutations in the transcription factor CCAAT-enhancer binding protein alpha (CEBPA) with mutations in colony stimulating factor receptor 3 (CSF3R). Mutations in CEBPA occur in approximately 10% of AML where they block differentiation and convey favorable risk. In contrast, CSF3R mutations lead to constitutive receptor activation and uncontrolled neutrophil proliferation. In the absence of co-occurring Class II mutations, membrane proximal CSF3R mutations produce the myeloproliferative neoplasm chronic neutrophilic leukemia (CNL). Interestingly, patients with CEBPA mutant AML that also harbor an oncogenic CSF3R mutation have worse prognosis than those with wild type CSF3R. However, the mechanism underlying this oncogene synergy remains unknown. To model the co-occurrence of these mutations, we expressed CSF3RT618I (The most common membrane proximal CSF3R mutation) in fetal liver hematopoietic stem cells harboring compound heterozygous CEBPA mutations in the endogenous allele (CEBPAK/L). Mice transplanted with mutant CEBPA alone developed a long latency AML with a median survival of 60 weeks. In contrast, mice transplanted with mutant CSF3RT618I/CEBPAK/L cells developed a much more rapid AML with a median survival of 13 weeks. These results were corroborated in an orthogonal model in which mutant CSF3R and a C-terminal mutant CEBPA were retrovirally expressed prior to bone marrow transplant. To dissect the underlying mechanism, we performed a comprehensive transcriptomic and epigenetic analysis on cells expressing each mutation in isolation as well as the combination. This analysis revealed that mutant CSF3R activates a distinct set of enhancers that regulate genes associated with differentiation and drive neutrophil differentiation. Co-expression of mutant CEBPA blocks the activation differentiation-associated enhancers but is permissive to those associated with proliferation. Differentiation but not proliferation-associated enhancers are bound by wild type CEBPA. Thus, the dominant negative impact of mutant CEBPA at these enhancers explains its differential impact on differentiative and proliferative transcriptional programs. Enhancer activation precedes promoter activation and CEBPA mutations are thought to represent early events in AML initiation. The epigenetic mechanism underlying the observed oncogene synergy argues that CEBPA mutations must occur prior to CSF3R to impact differentiation. We therefore developed a retroviral vector system enabling temporal control of Cre-mediated oncogene expression. Using this system, we found that only when mutant CEBPA is expressed prior to mutant CEBPA is differentiation arrest observed. Furthermore, AML develops in vivo only when mutant CEBPA is expressed prior to mutant CSF3R. To develop novel therapeutic strategies for this subclass of AML with adverse prognosis, we performed medium throughput drug screening on CSF3R/CEBPA mutant AML cells and identified sensitivity to inhibitors of JAK/STAT signaling as well as Lysine Demethylase 1 (LSD1). In other subtypes of AML, LSD1 inhibitors activate enhancers associated with differentiation. We confirmed that LSD1 inhibition promotes neutrophilic differentiation in CSF3R/CEBPA and through epigenetic and transcription profiling establish that this occurs via the reactivation of differentiation-associated enhancers. We further found that the combination of ruxolitinib (JAK/STAT inhibitor) and GSK2879552 produce a complete hematologic response and double median survival in mice harboring CSF3R/CEBPA mutant AML. Thus, the combination of JAK/STAT and LSD1 inhibitors represents and exciting therapeutic strategy for CSF3R/CEBPA mutant AML. Disclosures Druker: Celgene: Consultancy; Gilead Sciences: Other: former member of Scientific Advisory Board; ICON: Other: Scientific Founder of Molecular MD, which was acquired by ICON in Feb. 2019; Monojul: Other: former consultant; Novartis: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Patents & Royalties: Patent 6958335, Treatment of Gastrointestinal Stromal Tumors, exclusively licensed to Novartis, Research Funding; Bristol-Myers Squibb: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Research Funding; Pfizer: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Research Funding; Beat AML LLC: Other: Service on joint steering committee; The RUNX1 Research Program: Membership on an entity's Board of Directors or advisory committees; Patient True Talk: Consultancy; GRAIL: Equity Ownership, Other: former member of Scientific Advisory Board; Cepheid: Consultancy, Honoraria; Burroughs Wellcome Fund: Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Beta Cat: Membership on an entity's Board of Directors or advisory committees, Other: Stock options; Aptose Biosciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Amgen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; ALLCRON: Membership on an entity's Board of Directors or advisory committees; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees, Other: Stock options; OHSU (licensing fees): Patents & Royalties: #2573, Constructs and cell lines harboring various mutations in TNK2 and PTPN11, licensing fees ; Merck & Co: Patents & Royalties: Dana-Farber Cancer Institute license #2063, Monoclonal antiphosphotyrosine antibody 4G10, exclusive commercial license to Merck & Co; Dana-Farber Cancer Institute (antibody royalty): Patents & Royalties: #2524, antibody royalty; CureOne: Membership on an entity's Board of Directors or advisory committees; Pfizer: Research Funding; Aileron Therapeutics: #2573, Constructs and cell lines harboring various mutations in TNK2 and PTPN11, licensing fees , Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Patents & Royalties, Research Funding.

Improved Regulatory T Cell Activity in Patients with Chronic Immune Thrombocytopenia Purpura Treated with Thrombopoietic Agents.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 684-684 ◽  
Author(s):  
Weili Bao ◽  
Susanne Heck ◽  
Marissa Karpoff ◽  
James B. Bussel ◽  
Karina Yazdanbakhsh
Keyword(s):  
Board Of Directors ◽  
Immune Tolerance ◽  
Research Funding ◽  
Advisory Committees ◽  
Th Cells ◽  
Platelet Production ◽  
Platelet Counts ◽  
Treg Function ◽  
Equity Ownership ◽  
Pre Treatment

Abstract Abstract 684 Immune thrombocytopenic purpura (ITP) is an autoimmune, autoantibody-mediated disease with accelerated platelet destruction and impaired platelet production resulting in. thrombocytopenia and bleeding due to breakdown of immune tolerance. We and others have reported impaired regulatory CD4+CD25hi T cells (Treg) suppressive function in ITP patients. Clinical trials using thrombopoietic agents to stimulate platelet production have shown favorable outcomes in ITP patients with increased platelet counts and reduced bleeding. Two such agents, romiplostim (Nplate) and eltrombopag (Promacta), were recently licensed in the USA and elsewhere. Information on the immune responses of patients treated with these drugs pertaining to prognosis and response to treatment are lacking. We therefore studied the immunological profile of ITP patients with an average platelet counts of 21×109/L before treatment (n=6) and those who had been more than 3 months on treatment (total n=12, average platelet count 133 ×109/L) with either Nplate (n=5), eltrombopag (n=2) or an investigational thrombopoietic agent AKR-501 (n=5). We found no statistically significant differences in the Treg frequencies (Foxp3+CD25hi in the CD4+ population) of patients pre- and on-treatment (2.4± 0.6% versus 2.9± 0.5, p=0.5). However, the Treg activity as measured by suppression of proliferation of autologous CD4+CD25 cells at 1:1 and 1:4 ratios of Tregs : CD4+CD25 cells was significantly improved in patients on treatment compared to the pre-treatment group (at 1:1 ratio, 60% on-treatment versus 41% pre-treatment, p=0.001 and at 1:4 ratio, 44% versus 24%, p=0.04) and comparable to the overall Treg activity of controls (p=0.9). Improved Treg function correlated with reduction in IL-2-producing Th cells (Pearsons r=0.6, p=0.01), and patients on treatment had a 30% reduced frequency single positive type 1 IFN-g-producing Th cells (p=0.04). Circulatory levels of pro-inflammatory sCD40L in platelet poor plasma (PPP) were decreased in patients on treatment versus pre-treatment (0.54 ± 0.05 ng/ml versus 0.89 ± 0.13ng/ml, p=0.02) and comparable to controls (p=0.9). TGF-b levels in PPP strongly correlated with improved platelet counts (Pearsons r=0.8, p<0.0001) and were increased in patients following treatment (2177 ± 258 ng/ml versus 1083 ± 127 ng/ml p=0.004). In summary, our data indicates that treatment with thrombopoietic agents results in improved immune regulation by Tregs which may be responsible for the observed dampening of the pro-inflammatory Th1-associated responses. The concomitant increase in TGF-b levels and decrease in sCD40L levels, both of which are platelet-derived, are consistent with a rise in platelet counts in patients on treatment. This raises the interesting possibility that platelets in patients on treatment may play a role in improving Treg function either directly through cell-cell interactions or indirectly by release of TGF-b. In conclusion, our findings suggest that thrombopoietic agents are not immunologically inert but rather have profound effects to restore immune tolerance. Disclosures: Bussel: Genzyme: Research Funding; GlaxoSmithKline: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cangene: Research Funding; Amgen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Immunomedics: Research Funding; Ligand: Membership on an entity's Board of Directors or advisory committees, Research Funding; Eisai, Inc: Research Funding; Sysmex: Research Funding; Scienta: Speakers Bureau; Shionogi: Membership on an entity's Board of Directors or advisory committees.

Targeting Ribonucleotide Reductase M1 and M2 in Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 360-360
Author(s):  
Morihiko Sagawa ◽  
Hiroto Ohguchi ◽  
Takeshi Harada ◽  
Mehmet K. Samur ◽  
Yu-Tzu Tai ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Cell Lines ◽  
Plasma Cells ◽  
Small Cell ◽  
Wild Type ◽  
Small Cell Lung ◽  
Advisory Committees ◽  
Dna Damaging Agents ◽  
Equity Ownership

Abstract Ribonucleotide reductase (RR) is an enzyme that catalyzes the conversion of ribonucleotide diphosphate to deoxyribonucleotide diphosphate and is essential for de novo DNA synthesis, DNA repair, and cell growth. RR primarily exists as a heterodimer tetramer composed of regulatory subunit RRM1, and catalytic subunit RRM2. Overexpression or polymorphisms of RR are described non-small cell lung, pancreas, breast, and ovarian cancers. Moreover, RRM1 and RRM2 expression is highly correlated with patient survival in non-small cell lung and pancreas cancers. To date, the biologic significance in multiple myeloma (MM) has not yet been elucidated. In this study, we characterized the role of RRM1 and RRM2 in MM pathogenesis. By examining 3 independent expression data from GEO database, we found that RR (especially RRM1) expression is higher in MM plasma cells than normal plasma cells. We examined RRM1 and RRM2 expression in 6 MM cell lines and 3 MM patients' tumor cells by quantitative real-time PCR and immunoblotting and confirmed that both RRM1 and RRM2 are highly expressed in these cells. We next knocked down both RRM1 and RRM2 in 4 MM cell lines (NCI-H929, MM.1S, RPMI8226, and KMS-11) using siRNA. Knockdown of RRM1 and RRM2 triggered significant growth inhibition, associated with apoptotic cells death evidenced by AnnexinV/PI staining, in NCI-H929 and MM.1S, but not RPMI8226 or KMS-11 cells. We next examined molecular mechanisms whereby RRM1 downregulation triggers MM cell death. Gene expression profiling showed that p53 regulated genes were overexpressed after RRM1 knockdown. We therefore further examined DNA damage response (phosphorylated-ATM, -ATR, -Chk1, and -Chk2, and gamma-Histone H2A.X) and p53 (phosphorylated-p53, p21, Noxa, Puma, Bax) signaling pathways and found that these pathways were activated in NCI-H929 and MM.1S (both p53 wild-type), but not in RPMI8226 (p53 mutant) or KMS-11 (p53 null) cells after RRM1 knockdown. To validate the role of RRM1 of in vivo, we subcutaneously injected MM.1S cells transduced with shRNA against RRM1 or shLuc into our mouse xenograft model and observed that tumor growth was significantly reduced in shRRM1-MM.1S cells versus shLuc-MM.1S cells. Clofarabine (CLO), a purine nucleoside analog, which allosterically inhibits both DNA polymerases and RRM1 is used to treat acute leukemia and chronic lymphocytic leukemia and has been studied preclinically in MM (Valdez et. al. Experimental Hematology 2013). We therefore next examined CLO as a potential therapeutic agent in MM. Consistent with RRM1 knockdown, CLO induced growth arrest in p53 wild-type cell lines (NCI-H929, MM.1S, and MOLP-8), but not in p53 mutant (RPMI8226, OPM2, U266) or null (KMS-11) cells. Moreover, CLO treatment combined with DNA damaging agents (Melphalan, Doxorubicin) triggered synergetic cell death in p53 wild-type MM cells. Our results therefore demonstrate that RR, especially RRM1, is a novel therapeutic target in patients with wild-type p53 MM, and provide the basis for clinical evaluation of CLO, alone or in combination with DNA damaging agents, to improve patient outcome. Disclosures Hideshima: Acetylon: Consultancy; C4 Therapeutics: Equity Ownership. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Oncoprep: Equity Ownership; Gilead: Membership on an entity's Board of Directors or advisory committees; Oncoprep: Equity Ownership; Gilead: Membership on an entity's Board of Directors or advisory committees; Acetylon: Equity Ownership; Celgene: Membership on an entity's Board of Directors or advisory committees; Acetylon: Equity Ownership; Millennuim: Membership on an entity's Board of Directors or advisory committees; Millennuim: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Equity Ownership; C4 Therapeutics: Equity Ownership; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Network Modeling Reveals CDC42BPA and CLEC11A As Novel Driver Genes of t(4; 14) Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 802-802 ◽  
Author(s):  
Deepak Perumal ◽  
Alessandro Lagana' ◽  
David Melnekoff ◽  
Ben Readhead ◽  
Brian Kidd ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Network Analysis ◽  
Cell Viability ◽  
Board Of Directors ◽  
Research Funding ◽  
Hub Genes ◽  
Driver Genes ◽  
Advisory Committees ◽  
Equity Ownership

Abstract Multiple Myeloma (MM) is a genetically complex malignancy arising from bone marrow plasma cells with 30,000 new cases reported every year in the US. It is characterized by heterogeneity in clinical presentation and response to treatment. Next generation sequencing (NGS) technologies have enabled a deeper insight into cancer genomes and transcriptomes at an unprecedented level of detail. MMRF CoMMpass is a longitudinal, prospective observational study that aims to collect and analyze sequencing and clinical data from >1,000 MM patients at initial diagnosis and at relapse. Such effort providesa unique opportunity to improve our knowledge of MM pathogenesis and identify novel mechanisms that drive disease progression as well as genomic aberrations that underlie them. We have developed MMnet, an integrative network model of MM based on 450 RNA-Seq samples from the IA7 release of CoMMpass. By using Weighted Gene Co-expression Network Analysis (WGCNA), we defined 37 modules of co-expressed genes, that were further characterized by functional enrichment analysis and correlation with genetic alterations inferred from Whole-Exome (WXS) and Whole-Genome data (WGS) and with clinical traits. For each module, we calculated intra-module connectivity to identify highly connected "hub" genes that represent likely control points for biological processes. The results of our analysis revealed several module hub genes that were not previously associated to MM. Specifically, CDC42BPA and CLEC11A were hub genes of a module strongly up-regulated in patients carrying t(4;14) translocation involving MMSET and FGFR3, and associated to rISS stage III and FGFR3 mutations. The t(4;14) translocation observed in ~15% of newly diagnosed MM patients is associated with very poor prognosis. This translocation was observed in 48/450 patients in the CoMMpass cohort (≈10%). MMSET and FGFR3 are considered oncogenic drivers in t(4;14) myeloma and are currently being investigated as therapeutic targets. Given the prognostic and therapeutic importance of these genes and the unknown role of hub genes in MM pathogenesis, we sought to determine the biological significance of CDC42BPA and CLEC11A and whether they played a regulatory role in t(4;14) myeloma. CDC42BPA encodes serine/threonine protein kinase MRCK, which is a downstream effector of CDC42, a protein involved in cell cycle regulation. CLEC11A is a member of the C-type lectin superfamily, which includes several genes responsible for modulation of specific immune response to pathogens in dendritic cells and is involved in cell adhesion and cell communication. We selected two MM cell lines expressing MMSET and whose profile was concordant with module activation: KMS-11 and KMS-26. KMS-11 also carried t(4;14) and an activating mutation in FGFR3 (Y373C). We depleted CDC42BPA and CLEC11A by transient transfection of siRNA and assayed protein levels as well as cell viability. In both cell lines, western blot confirmed depletion of siRNA target proteins and revealed decreased expression of MMSET and its interactor NFkB as compared to scrambled controls. Cell viability (CellTitre Blue) assay showed a decrease in the number of viable cells by 60% at 72h following depletion of CLEC11A and by 55% at 72h following depletion of CDC42BPA as compared to control cells. As knockdown of MMSET was previously reported to induce apoptosis in MM cells, we next asked whether knockdown of CDC42BPA and CLEC11A had a similar impact. There was a significant increase of about 30-60% in the fraction of Annexin V-positive cells in siRNA-transfected cells compared with controls, consistent with the induction of apoptosis as examined by AnnexinV/PI staining. These results confirmed the central role of CDC42BPA and CLEC11A as potential regulators of MMSET in MM cell survival and regulation. These genes are therefore novel drug targets for treating t(4;14) myeloma. Future investigations will aim at determining the biological mechanisms by which CDC42BPA and CLEC11A regulate MMSET. In summary, our network analysis of the CoMMpass dataset uncovered novel and complex patterns of genomic perturbation, specifically novel key driver genes of myeloma network modules with further implications for identification and targeting of hub genes in gene co-expression networks. Disclosures Chari: Amgen Inc.: Honoraria, Research Funding; Pharmacyclics: Research Funding; Array Biopharma: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Janssen: Consultancy, Research Funding. Cho:Genentech Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Agenus, Inc.: Research Funding; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Ludwig Institute for Cancer Research: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Research Funding. Barlogie:Signal Genetics: Patents & Royalties. Jagannath:Novartis: Consultancy; Merck: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Bristol-Myers Squibb: Consultancy. Dudley:Janssen Pharmaceuticals, Inc.: Consultancy; AstraZeneca: Speakers Bureau; NuMedii, Inc.: Equity Ownership; Ayasdi, Inc.: Equity Ownership; Ecoeos, Inc.: Equity Ownership; Ontomics, Inc.: Equity Ownership; NuMedii, Inc.: Patents & Royalties; Personalis: Patents & Royalties; GlaxoSmithKline: Consultancy.

scholarly journals Completely Non-Myeloablative/Non-Lymphoablative Conditioning for BMT/HSCT Using Anti-Ckit Immunotoxins

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 493-493 ◽  
Author(s):  
Agnieszka Czechowicz ◽  
Rahul Palchaudhuri ◽  
Amelia Scheck ◽  
Jonathan Hoggatt ◽  
Borja Saez ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Board Of Directors ◽  
Research Funding ◽  
Host Immunity ◽  
Solid Organ ◽  
Wild Type ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Conditioning Regimens ◽  
Equity Ownership

Abstract Bone marrow/hematopoietic stem cell transplantation (BMT/HSCT) holds the remarkable ability to correct any blood or immune disease. Unfortunately, despite the tremendous potential of this procedure, BMT remains fairly limited in part due to the severe risks associated with the toxic conditioning regimens, such as irradiation and chemotherapy that are currently employed to enable donor HSC engraftment. Although significant work has been done to dose reduce the amount of these preparative agents, patients still experience many side effects including neutropenia/infections, anemia, mucositis, infertility, organ damage and secondary malignancies. Complete elimination of these toxic conditioning regimens could dramatically improve the safety profile of BMT and expand the potential applications to include many more non-malignant hematologic disorders, a wide variety of autoimmune disorders including diabetes, as well as facilitate solid organ tolerance. We have previously shown that competition with host HSC limits donor HSC engraftment, and that in immunocompromised hosts antagonistic anti-ckit monoclonal antibodies deplete host HSC and are an effective and safe alternative conditioning approach (Czechowicz, Science 2007). However, this modality of conditioning is not effective in hosts with competent immune systems. To further understand efficacy of antagonistic anti-ckit conditioning, we tested its functionality in multiple strains of immunocompromised mice and show that inhibition of SCF signaling is not sufficient to deplete host HSC in mouse strains with competent B-cells or T-cells, and that the addition of these cells interferes with the ability of antagonistic anti-ckit antibodies to effectively condition. In an attempt to overcome this hurdle, wildtype mice were immune-depleted with a variety of regimens but none enabled antagonistic anti-ckit conditioning in the immunocompetent setting. To strengthen the potency of anti-ckit mAbs we linked them to protein synthesis toxins, which when internalized by host HSC led to their rapid decline in vitro and in vivo. Administration of anti-ckit-saporin to wild-type mice resulted in >99% depletion of host HSC (Ckit+Lin-Sca1+CD150+CD48-), and lack of residual host HSC activity in the bone marrow was confirmed by CFC assays and competitive transplantation into lethally irradiated recipients. Interestingly, although ckit is expressed by a majority of HSPC, LT-HSC were most significantly affected and no cellularity changes in the bone marrow were observed. Uniquely this regimen was entirely non-peripheral blood ablative unlike other more broadly targeted conditioning regimens such as CD45 immunotoxins (Palchaudhuri, Nat Biotech 2016), and treated animals did not experience any significant depletion of myeloid, lymphoid, or erythroid cells. Figure 1 Figure 1. Treatment with anti-ckit-saporin effectively conditioned wild-type animals and near complete donor granulocyte chimerism was rapidly achieved post transplantation of whole bone marrow cells (99.54 ± 0.35 % vs. 6.79 ± 0.57 %, p<0.001), a >25-fold increase compared to unconditioned controls. Similarly, anti-ckit-saporin conditioning enable efficient engraftment of FACS purified donor HSC (Ckit+Lin-Sca1+CD150+CD48-). In both settings, donor HSC chimerism matched donor granulocyte chimerism further confirming replacement of host HSC. Importantly, host immunity was entirely intact in these animals throughout, with slower recalibration of the longer-lived immune cells given the lack of their direct depletion. Figure 2 Figure 2. This work sets the stage for redefining the way BMT/HSCT is performed, as it opens up the possibility for entirely safe, quick and easy transplantation that potentially could be done in the outpatient setting with no perturbation to host immunity. Extrapolation of these methods to humans may enable efficient yet gentle conditioning regimens for transplantation, which is especially exciting in the gene-therapy settings where no immune suppression is required, allowing for simple, safe and curative treatment of a wide magnitude of grievous blood and immune diseases ranging from sickle cell to hemophilia to HIV. As multiple anti-ckit mAbs are currently in development and being tested in clinical trials, such an approach may be rapidly translatable to patients. Disclosures Czechowicz: Third Rock Ventures: Consultancy; Global Blood Therapeutics: Equity Ownership; Editas Medicines: Equity Ownership, Patents & Royalties; Decibel Therapeutics: Equity Ownership; Magenta Therapeutics: Consultancy, Equity Ownership, Patents & Royalties; Forty Seven Inc: Patents & Royalties. Palchaudhuri:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Hoggatt:Magenta Therapeutics: Consultancy, Equity Ownership, Research Funding. Scadden:Teva: Consultancy; Apotex: Consultancy; Magenta Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Dr. Reddy's: Consultancy; GlaxoSmithKline: Research Funding; Fate Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Bone Therapeutics: Consultancy. Rossi:Magenta Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Intellia Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Moderna Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Sign in / Sign up

Export Citation Format

Share Document