Exploiting Transcription-Replication Conflicts As a Novel Therapeutic Intervention in Multiple Myeloma

Abstract Multiple myeloma (MM) is the second most frequent hematological malignancy, characterized by the accumulation of malignant plasma cells (PCs) within the bone marrow. To date, there is no definitive treatment for this pathology and a majority of patients will invariably relapse. Antibody secretion, the key biological function of PCs, is maintained in malignant PCs meaning that these cells display an elevated transcriptional stress. Besides, malignant PCs face oncogene-induced replication stress concomitantly with cell cycle deregulation. Consequently, transcription and replication in malignant PCs need to be tightly coordinated to avoid too much interferences that would increase replication stress and genomic instability. A failure to cope with these transcription/replication conflicts (TRCs) could have a significant impact on mutagenesis involved in MM development. Importantly, these effects might open the therapeutic possibility of TRCs enhancement to specifically kill malignant PCs. Based on these observations, we identified a signature of 13 TRCs resolution factors significantly overexpressed in MM patients. Considering the potent role of TRCs resolution in MM cell adaptation to replication stress, we sought to identify the TRCs resolution factors that are associated with a poor outcome in MM. High expression of 9 out of the 13 TRCs resolution factors significantly overexpressed in malignant PCs are associated with a poor outcome in MM (TT2 cohort, n = 345). We gathered the prognostic value of these 9 genes within a Gene Expression Profile (GEP)-based TRC resolution score (TRC score). High TRC score is associated with a poor outcome in two independent cohorts of newly diagnosed MM patients treated by high dose therapy and autologous stem cell transplantation (Arkansas, TT2 cohort, n = 345; CoMMpass cohort, n = 674) (Fig.1A). Interestingly, we investigated the link between the TRC score and the MM cells drug response using our collection of human myeloma cell lines (HMCLs), and identified that HMCLs with high TRC score values are significantly more sensitive to Panobinostat histone deacetylase inhibitor, currently used in MM treatment at relapse (n = 11, p value < 0.05). Histone acetylation has been shown to promote R-loop formation that constitutes obstacles to replication fork progression. Using primary MM cells from patients (n = 12) co-cultured with their bone marrow microenvironment, we found that a high TRC score value is associated with a higher toxicity of Panobinostat (p value < 0.01). Therefore, the TRC score allows the identification of a MM patients subgroup with a poor outcome that could benefit from Panobinostat treatment. Interestingly, TRCs are promoted by R-loop formation and G-quadruplex (G4) stabilizers treatment. R-loops are formed by the reannealing of the nascent RNA with the template DNA (called an RNA:DNA hybrid). G4s are four-stranded secondary DNA structures, constituted of stacked guanine tetrads. Both structures are formed during transcription in G-rich DNA regions and can represent a barrier for replication fork progression if unscheduled. G4s can stabilize R-loops which have been shown to mediate DNA damage induced by G4 stabilizers. Interestingly, treatment with the G4 stabilizer Pyridostatin (PDS) was associated with significant toxicity on HMCLs (n = 15) (Fig.1B), and on primary MM cells of patients cocultured with their bone marrow microenvironment (n = 5, p value < 0.05). Interestingly, the combination of PDS and Panobinostat has a synergistic effect in HMCLs. We also found a correlation between HMCLs TRC score and the response to two Bromodomain and Extra-Terminal motif (BET) proteins inhibitors, I-BET-762 and RVX-208. The synergistic effect of PDS combination with I-BET-762 was validated in vitro. BET proteins inhibition has been shown to increase R-loop formation and DNA damage. Furthermore, we used inducible RNase H expression in HMCLs to specifically degrade RNA:DNA hybrids. RNase H expression resulted in a significant reduction of DNA damage response after PDS treatment (Fig.1C). Our results underline that spontaneous replication stress and genomic instability are related to R-loop formation and TRCs in MM cells. Altogether, these results emphasize the therapeutic potential of TRCs targeting in MM using G4 stabilizers alone or in combination with current treatments. Figure 1 Figure 1. Disclosures Vincent: Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees. Cartron: Roche, Celgene-BMS: Consultancy; Danofi, Gilead, Novartis, Jansen, Roche, Celgene-BMS, Abbvie, Takeda: Honoraria. Herbaux: Roche: Honoraria; Janssen: Honoraria; Takeda: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding. Moreaux: Diag2Tec: Consultancy.

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Higher Expressions of PTH Receptor Type 1 and/or 2 in Bone Marrow Is Associated to Longer Survival in Newly Diagnosed Myeloma Patients Enrolled in Total Therapy 3

Blood ◽

10.1182/blood.v124.21.3409.3409 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3409-3409 ◽

Cited By ~ 1

Author(s):

Maurizio Zangari ◽

Caleb K Stein ◽

Shmuel Yaccoby ◽

Donghoon Yoon ◽

Christoph Heuck ◽

...

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Bone Marrow ◽

Board Of Directors ◽

P Value ◽

Newly Diagnosed ◽

Advisory Committees ◽

Logrank Test ◽

Total Therapy

Abstract Higher Expressions of PTH Receptor Type 1 and/or 2 in Bone Marrow is Associated to Longer Survival in Newly Diagnosed Myeloma Patients Enrolled in Total Therapy 3 INTRODUCTION: The Total Therapy 3 enrolled 303 newly diagnosed multiple myeloma patients at Myeloma Institute for Research and Therapy. Protocol included 2 cycles of VTD-PACE (bortezomib, thalidomide, and dexamethasone and 4-day continuous infusions of cisplatin, doxorubicin, cyclophosphamide, and etoposide) as induction and consolidation therapy after melphalan-based tandem transplantation, which is followed by 3 years of intended maintenance with VTD in year 1 and thalidomide/dexamethasone in years 2 and 3. As part of the protocol, gene expression profiling was performed from baseline bone marrow biopsy samples in 178 individuals. We have previously reported the clinical correlation between response to bortezomib and serum parathyroid hormone variations in myeloma patients as well as the interaction between receptor 1 and proteasome inhibitors function in cell line and myeloma mouse model. In this study we examine the PTH receptor 1 and 2 expression levels and their correlation to survival in total therapy 3 enrolled patients. METHOD: Gene expression profiling was performed using Affymetrix U133 plus 2.0 Microarrays (Santa Clara, CA) in baseline bone marrow biopsy samples from 178 patients enrolled on total therapy 3. Of these 178 patients, 108 were male. The overall median age of these patients was 59 years old at enrollment; 10 % of patients were considered to have high risk disease by 70 GEP model. Cox proportional hazards analysis was performed on the MAS5 normalized log 2 expression values of PTH1R and PTH2R using overall survival as the end point. Optimal dichotomous break points were found for PTH1R and PTH2R that corresponded to the maximum log rank test statistic from all cox proportional hazard models examined. To confirm PTH receptor expression in bone marrow, we performed real-time PCR using Taqman probes (PTH1R: Assay ID Hs00174895_m1 and PTH2R: Assay ID Hs00175044_m1) on subset of samples. RESULTS: Based on cox proportional hazards regression of PTH1R and PTH2R expression values, patients with higher PTH1R and PTH2R expression demonstrated better survival compared to lower expressing patients. PTH1R expression above optimal break point of 8.92 had a hazard ratio of 0.583 with a 95% confidence interval of (0.351, 0.969) and logrank test p-value of 0.035. PTH2R expression above optimal break point of 6.85 had a hazard ratio of 0.541 with a 95% confidence interval of (0.323, 0.905) and logrank test p-value of 0.018. Furthermore, the patients that were lower expressed in both PTH1R and PTH2R performed significantly poorer in outcome (n= 24 and median survival of 4.52 years logrank p-value+5.71e-05). Real-time PCR using Taqman probes was able to demonstrate relatively high levels of PTH1R and PTHR2 transcripts at bone marrow level. Figure 1 Figure 1. CONCLUSIONS: This is the first report indicating that PTH receptors type 1 and 2 gene expression levels are positively associated to overall survival in symptomatic multiple myeloma patients. Also we describe the presence of PTH2R at bone marrow level which function appear associated to myeloma control. These data confirm the correlation and close interaction between the survival of multiple myeloma patients and the parathyroid hormone axis. Disclosures Zangari: Norvartis: Membership on an entity's Board of Directors or advisory committees; Onyx: Research Funding; Millennium: Research Funding. Heuck:Celgene: Honoraria; Foundation Medicine: Honoraria; Millennium: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees. van Rhee:Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees. Morgan:Celgene Corp: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Myeloma UK: Membership on an entity's Board of Directors or advisory committees; International Myeloma Foundation: Membership on an entity's Board of Directors or advisory committees; The Binding Site: Membership on an entity's Board of Directors or advisory committees; MMRF: Membership on an entity's Board of Directors or advisory committees.

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A Specific Mesenchymal Stem and Progenitor Cell (MSPC) Subpopulation with a Multi-Potent Gene Signature Is Transcriptionally Altered in the Setting of Myelodysplastic Syndrome (MDS) in Primary Human Bone Marrow Aspirates

Blood ◽

10.1182/blood-2019-130227 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1708-1708

Author(s):

Thomas J Fountaine ◽

Mark W LaMere ◽

Daniel K Byun ◽

Jason R Myers ◽

John M Ashton ◽

...

Keyword(s):

Bone Marrow ◽

Board Of Directors ◽

Surface Expression ◽

Cell Surface Expression ◽

P Value ◽

Risk Category ◽

Gene Set Enrichment ◽

Advisory Committees ◽

Gene Sets ◽

Equity Ownership

Introduction: Myelodysplastic syndromes (MDS) are genetically diverse and heterogeneous diseases characterized by dysplasia and cytopenias and a dismal prognosis of ˂ 50% overall survival at 5-years. In vitro and in vivo experimental data have shown that the bone marrow microenvironment (BMME) may play a role in disease progression and, importantly, in murine models, transplantation of MDS to a healthy BMME was shown to mitigate disease. Mesenchymal Stem and Progenitor Cells (MSPCs), as part of the BMME, give rise to multiple non-hematopoietic progenitor cells and provide essential support to hematopoietic stem cells. MSPCs have also been shown to be functionally altered in patients with myeloid neoplasias. Murine studies have demonstrated distinct roles for specific subsets of bone marrow mesenchymal cells in myeloid malignancies. We hypothesized that specific subsets of human bone marrow MSPCs will play differential roles in the pathogenesis of MDS. Using flow-cytometry, high-throughput sequencing and gene set enrichment analysis (GSEA), we characterized human MSPCs in the bone marrow aspirates from patients with MDS and normal healthy controls (NBM). Methods: Mononuclear cells were isolated by iliac crest bone marrow aspiration from 10-healthy donors and 14-patients at the University of Rochester Medical Center. Within the non-hematopoietic (CD45-, CD235-), non-endothelial (CD31-) bone marrow compartment, we enriched and isolated 3 distinct-subpopulations of MSPCs based on cell-surface expression of CD271/NGFR, CD106/VCAM-1 and CD146/MCAM. Populations were defined as follows: CD271+/CD146- (CD271+), CD271+/CD146+/CD106+ (DPCD106+), and CD271+/CD146+/CD106- (DPCD106-). RNA-seq analysis was performed on each subpopulation to define transcriptional signatures (TS) and gene set enrichment patterns. Statistically significant differentially expressed genes (DEGs) were defined by fold-change ≥ ±1 and p-value ˂0.05. Results: We first set out to define differences in the TS and interrogate the function of MSPC populations in NBM. Principle component analysis (PCA) demonstrated the highest variance between the DPCD106+ and the CD271+ populations. The number of DEGs were also highest between the DPCD106+ and CD271+ populations (n=3,619 genes). GSEA identified 745 and 336 gene sets with positive enrichment in the DPCD106+ and CD271+ group, respectively, and illustrated that the DPCD106+ population was significantly enriched in gene sets involved in early embryonic developmental and "stem-like" pathways whereas the CD271+ population was enriched in cell cycling, DNA and chromosomal organization. In the setting of MDS, the mean relative frequency of MDS CD271+ nearly tripled (0.4230/0.1445; p-value 0.045). Compared to NBM, MDS DPCD106+ cells had the highest variance by PCA and the highest number of DEGs (n=560). GSEA identified 19-gene sets with significant enrichment in the MDS DPCD106+ group and, intriguingly, 12 (63%) were identical to gene sets enriched in the CD271+ group. Furthermore, of the 560 DEGs in the MDS DPCD106+ MSPCs, 300 were upregulated and, of those, 160 (53%) were identical to upregulated genes in the CD271+ NBM group including the acquisition of a proliferative signature. Altogether, this data suggests a switch in the TS of theDPCD106+ population in the setting of MDS. Importantly, this TS clustered MDS DPCD106+ from NBM, regardless of MDS risk category. Conclusion: We successfully characterized 3 subtypes of MSPCs in NBM and MDS. In NBM, we demonstrate that cell surface expression of CD271, CD146 and CD106 defined the most stem-like TS within the non-hematopoietic, non-endothelial bone marrow compartment. In the setting of MDS, the increase in population frequency of the CD271+ cells and the concomitant transcriptomic aberrations observed in the MDS derived DPCD106+ population support the hypothesis that specific MSPC populations have differential roles in MDS pathogenesis. Further, we identify a TS that discriminates MDS derived MSPCs from NBM irrespective of MDS-risk category. This suggests that alterations within specific MSPC populations may represent a unifying pathway in disease pathogenesis despite heterogeneity and genetic drivers intrinsic to the MDS clone. Thus, targeting the BMME represents a potentially novel therapeutic strategy aimed at mitigating disease and restoring normal hematopoiesis in patients with MDS. Disclosures Liesveld: Onconova: Other: Data safety monitoring board; Abbvie: Membership on an entity's Board of Directors or advisory committees. Scadden:Editas Medicine: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Bone Therapeutics: Consultancy; Clear Creek Bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Novartis: Other: Sponsored research; Fate Therapeutics: Consultancy, Equity Ownership; Red Oak Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Fog Pharma: Consultancy; Agios Pharmaceuticals: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; LifeVaultBio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

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Impact and Function of Somatic PHF6 Mutations in Myeloid Neoplasms

Blood ◽

10.1182/blood.v124.21.3581.3581 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3581-3581

Author(s):

Brittney Dienes ◽

Bartlomiej P Przychodzen ◽

Michael Clemente ◽

Wenyi Shen ◽

Chantana Polprasert ◽

...

Keyword(s):

Bone Marrow ◽

Dna Damage ◽

Board Of Directors ◽

Somatic Mutations ◽

Suppressor Gene ◽

Advisory Committees ◽

Nonsense Mutations ◽

Dysmorphic Features ◽

Normal Bone ◽

Myeloid Neoplasms

Abstract Borjeson−Forssman−Lehmann syndrome (BFLS), a hereditary X-linked disorder characterized by mental retardation, truncal obesity, gynecomastia, hypogonadism and other dysmorphic features, is known to be caused by germline (GL) mutations of plant homeo domain finger protein 6 (PHF6). PHF6 is a highly conserved 41kDa protein showing ubiquitous expression in a variety of tissues, including bone marrow, CD34+ cells and blood leukocytes. Human PHF6 is located on chrXq26.2. Recently, rare somatic nonsense mutations and deletions have been detected in patients with T-ALL and AML and found in some T-ALL cell lines. Patients with BFLS with PHF6 mutations have been reported to develop leukemia, suggesting PHF6 mutations may predispose cancer. Although the actual function and molecular pathogenesis is unknown, PHF6 has been suggested to be a tumor suppressor gene involved in the control of myeloid development. In an index case of a young adult female patient with proliferative CMML with dysmorphic features, we have identified remarkable GL mosaicism for PHF6 mutation (p.K44fs), confirmed by deep sequencing of marrow, CD3+ cells and skin tissue. Subsequently, we screened patients with myeloid neoplasms by targeted multi-amplicon sequencing to determine the prevalence and distribution of PHF6 gene alterations. Sequencing results from 1072 cases were analyzed (728 by targeted deep sequencing and 344 by whole exome sequencing). In total, we identified 21 cases with PHF6 mutations, 13 of which were frameshift or nonsense mutations. Previously, PHF6 have been included in screening panels by Haferlach et al., (Leukemia 2014) and Papaemmanuil et al., (Blood 2013) and somatic mutations were found in 24/944 and 21/738 cases of MDS, respectively. These results along with ours suggest that PHF6 mutations are common driver events. The somatic nature of these defects was confirmed by analysis of non-clonal CD3+ lymphocytes, thus, PHF6 mutations occur at a frequency of 2.0% and are most frequently observed among patients with secondary AML (33%, P=.0021). Gender distribution showed a strong male predominance (76%), likely due to the location of PHF6 on chrX and indicating that retention of a single copy of PHF6 may be protective. SNP-array karyotyping showed that deletions of Xq, involving the PHF6 locus (Xq26), were present in about 1.2% of myeloid neoplasms and affect only female patients. As a family, plant homeo domain (PHD) finger genes are affected by mutations associated with various cancers. JARID1A, PHF23, NSD1 and NSD3 were described to serve as fusion partners with the NUP98 in a subset of AML cases. The most frequent chromosomal aberration observed in conjunction with PHF6 mutations was trisomy-8 (P=.08). The most commonly associated somatic mutations were in RUNX1 (N=7; P=.001), U2AF1 (N=5), ASXL1 (N=5), IDH1 (N=4), and DNMT3A (N=4). Interestingly, 6/7 cases with concomitant PHF6 and RUNX1 mutations showed a poor prognosis AML. Subsequent analysis of clonal architecture using variant allelic frequency calculations and serial samples for these cases suggested that PHF6 may function as a founder driver gene while RUNX1 mutations are acquired as secondary events. Recent studies proposed that PHF6 deficiency leads to impaired cell proliferation, cell cycle arrest at G2/M phase and an increase of DNA damage. To examine DNA damage and quantify double stranded breaks (DSBs) in primary cells from PHF6-mutants, those with wild-type (WT) PHF6 and normal bone marrow we used a flow cytometric anti-γH2AX assay, following induction of DNA damage with Camptothecin. As judged by greater percentages of anti-γH2AX labeled cells, DSBs were more common in mutant cases consistent with more DNA damage present in PHF6 mutant compared to WT MDS and normal bone marrow cells. In conclusion, our results indicate that PHF6 mutations are generally present in more aggressive types of myeloid neoplasms, frequently associated with RUNX1 mutations. Our functional in vitro studies along with recently published reports suggest an association of PHF6 deficiency with genomic instability and thereby provide a basis for a mutator phenotype conveyed by ancestral lesions, consistent with its role as a tumor suppressor gene. Disclosures Sekeres: Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen Corp: Membership on an entity's Board of Directors or advisory committees; Boehringer-Ingelheim Corp: Membership on an entity's Board of Directors or advisory committees.

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Protection from UV-Light Is an Evolutionarily Conserved Feature of the Hematopoietic Niche

Blood ◽

10.1182/blood.v128.22.2663.2663 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2663-2663

Author(s):

Friedrich G Kapp ◽

Julie R Perlin ◽

Eirini Trompouki ◽

Charlotte M Niemeyer ◽

Wolfgang Driever ◽

...

Keyword(s):

Bone Marrow ◽

Dna Damage ◽

Protective Effect ◽

Board Of Directors ◽

Uv Light ◽

Advisory Committees ◽

Marrow Cavity ◽

Bone Marrow Cavity ◽

Equity Ownership

Abstract Hematopoietic stem and progenitor cells (HSPC) reside in the bone marrow cavity in all mammals, but other locations are used in non-mammalian animals, such as the kidney marrow in zebrafish. The niche location is thought to be selected based on unique advantages for HSPC function and protection in each organism. Studying the zebrafish HSPC niches during development and adulthood, we observed that melanocytes occupy the location above the kidney prior to the arrival of HSPCs. To elucidate whether these particular melanocytes play a role for HSPCs, we compared HSPCs in wildtype and nacre-mutant embryos (that lack all melanocytes) by cmyb in situ at different time-points. There were no obvious differences, suggesting that melanocytes are dispensable for steady state hematopoiesis. Since the position of the melanocytes above the HSPCs was very consistent in different strains and at various ages, we asked whether the melanocytes protect the HSPC niche from UV light. Indeed, after irradiation with UV light, levels of UV-induced DNA damage were approximately 50% higher in HSPCs isolated from kidneys of unpigmented embryos compared to their pigmented siblings as assessed by immunocytochemistry with a cyclobutane pyrimidine dimer antibody (TDM-2). In addition to acquiring more DNA damage, the unpigmented embryos also showed a significantly more pronounced decrease of HSPCs as assessed by cmyb in situ: 55% of control embryos had high cmyb staining. Upon irradiation 44% of irradiated pigmented embryos retained high c-myb staining compared to only 28% of irradiated unpigmented embryos (total embryos analyzed: n= 29, 27 and 36 respectively; p=0.01 between the latter two groups). The reduction of HSPCs could not be rescued in unpigmented p53-mutant embryos, indicating a p53-independent mechanism of HSPC loss after UV irradiation. The protective effect of melanocytes could either be due to a physical shielding of the HSPCs or due to a paracrine mechanism, such as secretion of cytokines or uptake of reactive oxygen species. The paracrine mechanism could be dependent on melanin. To address this question, we focused the path of light at a different angle in which the melanocytes would not be able to block the UV light from reaching the kidney marrow. This abrogated the protective effect of pigment containing melanocytes and caused a reduction of HSPCs by cmyb in situ to the levels of unpigmented siblings: pigmented embryos irradiated at an angle now also had high cmyb staining in only 28% of the embryos (n= 25). This suggests that the melanocytes only provide shade for the HSPCs. We then asked if this protective pigmentation pattern is specific to zebrafish and traced the origin of this trait by comparative histology. We found that other teleost fish but also evolutionarily older fish such as sturgeon and polypterus have melanocytes covering the kidney marrow. Even in lamprey, an ancient jawless vertebrate, the HSPC niche (located in the supraspinal organ) is covered by melanocytes, which allows us to date the evolutionary origin of UV protection back to at least 500 million years. We performed histological studies in frogs and found that the HSPCs reside under a melanocyte shield in the tadpole stage, while it is known that in adult frogs the HSPCs move into the bone marrow cavity. Considering that all terrestrial animals such as reptiles, birds and mammals host hematopoiesis in the bone marrow, we suggest that during the transition from aquatic to terrestrial life, the HSPCs were driven out of their melanocyte covered niche due to the high level of irradiation and relocated into a more protective niche, the bone marrow. Disclosures Zon: Scholar Rock: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Marauder Therapeutics: Equity Ownership, Other: Founder; Fate, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder.

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Characterization of Philadelphia-Negative Myeloproliferative Neoplasms By the Bone Marrow Immune Microenvironment

Blood ◽

10.1182/blood-2021-152341 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 4593-4593

Author(s):

Sung-Eun Lee ◽

Seowon Choi ◽

Gi June Min ◽

Sung-Soo Park ◽

Silvia Park ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

T Cell ◽

Board Of Directors ◽

Myeloproliferative Neoplasms ◽

Cd8 T Cell ◽

Fold Change ◽

P Value ◽

Immune Microenvironment ◽

Advisory Committees

Abstract Backgound The Philadelphia-negative myeloproliferative neoplasms (MPNs) share similar molecular characteristics, in which the excessive myeloproliferations are driven by mutations in JAK2, CALR, MPL, and uncommon variants. More recently, the biological basis on acquisition of somatic mutations have been accumulated. However, symptoms, histomorphologic characteristics, and natural histories are different between MPN subsets. Although there are increasing evidences that inflammation have a key role in promoting MPN initiation and influencing disease evolution, characteristics of the bone marrow immune microenvironment between MPN subsets remains unclear exactly. Aims To characterize the bone marrow immune microenvironment of Philadelphia-negative MPNs, we carried out immune-related gene expression profiling of bone marrow aspirates (BMAs) from 33 MPN patients (6 PV, 6 ET, 6 early PMF, and 15 overt MF including 10 primary MF, 5 post-PV/ET MF) using nCounter Immunology Panel. Methods BMA samples collected at diagnosis using EDTA-coated tubes. Those samples were processed within 24 hours from collection to obtain mononuclear cells by density centrifugation using Ficoll-Paque. NanoString analysis using a 594-gene nCounter Immunology panel (Human v2 - nanoString) was performed on RNAs extracted from 33 MPN bone marrow aspirates. Results First, to investigate whether there are distinct gene expression signatures of immune cells between three subcategories of MPNs, we compared gene expression profiles (GEPs) between ET, PV, and overt PMF. Using a P-value cutoff of ≤0.05 and fold-change ≥ 2, 10 upregulated and 32 downregulated differentially expressed genes (DEGs) were identified in ET than PMF, and 9 upregulated and 11 downregulated DEGs were identified in PV than PMF, while we found no significant DEGs between ET versus PV, except seven genes. Second, we investigated differences in GEPs between early PMF and overt PMF. Thirty-two downregulated and 4 upregulated DEGs were identified in early PMF than overt PMF. Gene set analysis revealed that the expression of genes related to almost processes decreased in early PMF than overt PMF. Then, we questioned differences between PMF and post-PV/ET MF. Using a P-value cutoff of ≤0.05 and fold-change ≥ 2, 12 upregulated and 12 downregulated DEGs were identified. Next, we computed relative abundances of immune cell subpopulations, estimated based on expression counts from the entire panel of surveyed genes, and compared them between the subcategories of MPNs. The abundance measurement of exhausted CD8 + T cell genes were significantly lower in ET and PV, compared with overt PMF, suggesting T cell exhaustion was distinct in overt PMF, compared to ET and PV. Conclusions The results demonstrated that immune microenvironment signature was distinguishable in the subcategories of MPNs. In addition, inflammatory signature was enriched in the bone marrow of overt PMF and exhausted CD8 + T cell genes were distinct in overt PMT. Further investigation is warranted to determine the immunological factors critical for potential therapeutic targets to alleviate progress to myelofibrosis. Disclosures Kim: AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; AIMS Biosciense: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; AML-Hub: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; BL & H: Research Funding; BMS & Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Boryung Pharm Co.: Consultancy; Daiichi Sankyo: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Handok: Consultancy, Honoraria; LG Chem: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria; Pintherapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi Genzyme: Honoraria, Speakers Bureau; SL VaxiGen: Consultancy, Honoraria; VigenCell: Consultancy, Honoraria. Lee: Alexion, AstraZeneca Rare Disease: Honoraria, Membership on an entity's Board of Directors or advisory committees.

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An Intergroup Randomised Trial of Rituximab Versus a Watch and Wait Strategy In Patients with Stage II, III, IV, Asymptomatic, Non-Bulky Follicular Lymphoma (Grades 1, 2 and 3a). A Preliminary Analysis

Blood ◽

10.1182/blood.v116.21.6.6 ◽

2010 ◽

Vol 116 (21) ◽

pp. 6-6 ◽

Cited By ~ 44

Author(s):

Kirit M Ardeshna ◽

Wendi Qian ◽

Paul Smith ◽

June Warden ◽

Lindsay Stevens ◽

...

Keyword(s):

Bone Marrow ◽

Follicular Lymphoma ◽

Board Of Directors ◽

Median Time ◽

Watchful Waiting ◽

Rank Test ◽

P Value ◽

Advisory Committees ◽

New Therapy ◽

Time To Initiation

Abstract Abstract 6 Patients with asymptomatic, advanced stage, follicular lymphoma have shown no benefit of immediate chemotherapy when compared with a watchful-waiting approach, whereby chemotherapy is deferred until disease progression. Deferring chemotherapy may spare the patient the side effects of the chemotherapy in the short term and historically this has been the preferred approach. With the advent of rituximab and its relatively favourable side effect profile we designed this study to compare a watchful waiting approach with immediate treatment with rituximab. Adult patients with asymptomatic stage 2, 3 or 4 follicular lymphoma (grades 1–2 & 3a) and adequate bone marrow reserve were randomly assigned with a ratio 1:1:1 to watchful waiting (arm A) or rituximab 375mg/m2 weekly for 4 weeks (arm B) or rituximab 375mg/m2 weekly for 4 weeks followed by rituximab maintenance every 2 months for 2 years (starting at month 3 until month 25)(arm C). The primary endpoints were a) time to initiation of new therapy (chemotherapy or radiotherapy) and b) effect on quality of life. The study was designed to detect an improvement in the median time to initiation of therapy in each of the rituximab arms of 18 months (from 30 months to 48 months) with 2.5% significance level and 90% power. A total of 230 events were required and 600 patients were planned. In September 2007, a decision was made to discontinue arm B as evidence of the efficacy of maintenance rituximab became clear. With the two arms comparison, using a significance level of 5%, a total of 360 patients in Arm A and Arm C were planned. Between September 2004 and May 2009 462 patients were randomised (186 Arm A, 84 Arm B, and 192 Arm C). 95% of patients had low tumour burden (GELF criteria) the other 5% had raised LDH but fulfilled the remaining GELF criteria. 98% were entered into the study within 4 months of diagnostic biopsy. Median age 60yr (range27-87). 54% female. ECOG performance status 0=91% & 1=9%. Grade 1–2=89%. Stage 2(21%), stage 3(40%), stage 4 (39%). 42% had bone marrow involvement. FLIPI score: 0=9%, 1=26%, 2=41%, 3=22%, 4=2%. In March 2010 the Data Monitoring committee concluded that the data regarding time to initiation of new therapy was mature and recommended full analysis of data to be performed and presented in the knowledge that rituximab maintenance was still ongoing in 20 patients. To date 45 SAEs have been reported (Arms A=14, B=6, C=25), 14 SAE were considered possibly, probably or definitely related to the study drug (Arm B=4, C=10). 5 allergic reactions (two grade 3), 6 infections, 3 episodes of grade 4 neutropenia. Responses were assessed at month 7, 13 and 25. CT was compulsory at months 7 and 25. Bone marrow was only required if CR on clinical and CT criteria. An interim analysis was performed on 9 Feb 2010. At month 7: Arm A: spontaneous remission was seen in 3%, PR=6%, NC (no change) =74%, PD=17%. Arm B: CR+CRu=45%, PR=33%, NC=19%, PD=3%. Arm C: CR+CRu=49%, PR=36%, NC=11%, PD=3%. At the time of the interim analysis 93 (20%) patients had initiated new treatment. Of these 93 patients 84 (90%) had clinically reported progression. New treatment was chemotherapy in 78 (84%), radiotherapy in 10 (11%), rituximab monotherapy in 2 (2%), surgery in 1(1%),currently not known in 2 (2%). The estimated median time to initiation of new therapy in arm A was 33 months, similar to our previous trial of watchful waiting (Ardeshna et al Lancet 2003). The time to initiation of new therapy was significantly longer in the rituximab arms (fig 1, p value of log-rank test <0.001 for each of rituximab arms vs arm A) and the median time was not reached at 4 years. Not all patients who were reported to have clinically progressed (n=142) warranted initiation of therapy (n=84). There were again significant differences in progression-free survival between the observation and rituximab arms (fig 2, p value of log-rank test <0.001 for each of rituximab arms vs arm A). 98% of patients remain alive and there was no different in overall survival between the 3 arms (p value >0.5). These data indicate that initial treatment with rituximab significantly delays the need for new therapy and this finding may change the management of patients with newly diagnosed asymptomatic follicular lymphoma. Disclosures: Ardeshna: Roche: Funding data manager, Honoraria, Membership on an entity's Board of Directors or advisory committees. Off Label Use: Rituximab is currently not licensed for use as a single agent in previously untreated patients with with asymptomatic advanced stage follicular lymphoma. Pocock:Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees. Cunningham:Roche: Research Funding. Davies:Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; chugai: Honoraria, Research Funding. Walewski:Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Linch:Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees.

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Germline Runx1 Mutations Induce Inflammation in Hematopoietic Stem and Progenitor Cells and Predispose to Hematologic Malignancies

Blood ◽

10.1182/blood-2021-153433 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2201-2201

Author(s):

Mohd Hafiz Ahmad ◽

Mahesh Hegde ◽

Waihay J. Wong ◽

Andrew Dunbar ◽

Anneliese Carrascoso ◽

...

Keyword(s):

Bone Marrow ◽

Dna Damage ◽

Progenitor Cells ◽

Board Of Directors ◽

Research Funding ◽

Bone Marrow Cells ◽

Hematologic Malignancies ◽

Advisory Committees ◽

Hematopoietic Stem

Abstract Patients with Familial Platelet disorder (FPD) have a germline RUNX1 mutation and are at high risk to developing hematologic malignancies (HM), primarily myelodysplastic syndrome and acute myeloid leukemia (lifetime risk~40%). To understand how germline RUNX1 mutations predispose to HM in vivo, we developed a Runx1 R188Q/+ mouse strain , mimicking the FPD-associated R201Q missense mutation. Analysis of the bone marrow cells in Runx1 R188Q/+ mice revealed a significant increase in the total number of bone marrow cells. Immunophenotypic analysis using Sca-1 and Cd86 markers revealed a significant increase in Sca-1 expression in hematopoietic stem and multi-potential progenitor cells, indicating a systemic inflammation in the bone marrow. In addition, the frequency of common-myeloid, granulocytic-monocytic and granulocytic progenitor cells were found significantly increased in the Runx1 R188Q/+ bone marrow. Accordingly, their colony-forming unit capacity was increased when compared to wildtype controls (wt/Runx1 R188Q/+ CFU average = 45/85), indicating a myeloid bias. The number and size of platelets were not altered in Runx1 R188Q/+ mice. However, platelet function was significantly reduced. The activation of the Cd41/Cd61 fibrinogen receptor complex in membrane after thrombin treatment was reduced in Runx1 R188Q/+ platelets. Similarly, the translocation of P-selectin by alpha granules and the secretion of serotonin by the dense granules were also reduced. Hematopoietic progenitor cells isolated from Runx1 R188Q/+ mice revealed a significant reduction in DNA-damage repair response in vitro. Quantitative analysis of nuclei with 53bp1-positive foci in response to ionizing radiation showed a marked increase in 53bp1-positive foci in Runx1 R188Q/+ nuclei, suggesting that Runx1 R188Q/+ cells have a defective repair of double strand DNA breaks. Furthermore, expression of DNA-damage repair pathway-associated Pmaip1 (Noxa) was significantly reduced in irradiated Runx1 R188Q/+ hematopoietic progenitor cells. To understand underlying mechanism responsible for the observed myeloid bias in Runx1 R188Q/+ cells, transcription profiling analysis was performed in myeloid progenitors from wildtype and Runx1 R188Q/+ mice, utilizing RNA-sequencing. A total of 39 genes were significantly deregulated (> 1.5 FC; FDR<0.05), including 8 up- and 31 down-regulated genes. The expression of three repressed genes with important function in hematopoietic differentiation and malignancy (Cdh1, Gja1, and Fcer1a) were validated by qRT-PCR. To study the FPD-associated pre-leukemic process in vivo, wildtype and Runx1 R188Q/+ mice were monitored for 20 months. Although Runx1 R188Q/+ mice remained healthy for 18 months, somatic mutations in their leukocytes were evident at 12 months. Targeted sequencing of 578 cancer genes (mIMPACT panel) in leukocyte DNA of two Runx1 R188Q/+ mice identified somatic mutations in Kdm6a, Setd1b, Amer1, and Esco1 (variant allele frequencies between 0.5% and 2.8%). These mutations were confirmed at stable frequency for eight following months. Since loss of the second Runx1 allele is a frequent somatic event in progression to FPD/HM, we evaluated the predisposition to HM in Mx1Cre-Runx1 R188Q/fl mice over time. Unlike Runx1 R188Q/+ mice, Runx1 R188Q/Δ mice succumbed to myeloid leukemia with a median latency of 37.5 weeks and full penetrance. In addition, the expression of oncogenic Nras-G12D, in Runx1 R188Q/Δ mice reduced the median latency to 14.7 weeks. These studies demonstrate that FPD-associated Runx1 germline mutations induce inflammation in hematopoietic stem cells, induce myeloid expansion with defective DNA-damage response and predispose to HM over time. These studies suggest that anti-inflammatory therapies in pre-symptomatic FPD patients may reduce clonal expansion and predisposition to HM. Disclosures Ebert: Exo Therapeutics: Membership on an entity's Board of Directors or advisory committees; Skyhawk Therapeutics: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Deerfield: Research Funding; GRAIL: Consultancy. Levine: Isoplexis: Membership on an entity's Board of Directors or advisory committees; Auron: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Zentalis: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; QIAGEN: Membership on an entity's Board of Directors or advisory committees; Ajax: Membership on an entity's Board of Directors or advisory committees; Imago: Membership on an entity's Board of Directors or advisory committees; Mission Bio: Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria; Prelude: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy; Lilly: Honoraria; Morphosys: Consultancy; Roche: Honoraria, Research Funding; Incyte: Consultancy; Astellas: Consultancy; Amgen: Honoraria.

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Donor Cell Acute Myeloid Leukemia after Transplant for Follicular Lymphoma

Blood ◽

10.1182/blood-2019-122932 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5697-5697

Author(s):

Lacey S. Williams ◽

Catherine E. Lai

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Follicular Lymphoma ◽

Board Of Directors ◽

Myeloid Leukemia ◽

Donor Cell ◽

Cell Leukemia ◽

Advisory Committees ◽

Donor Cell Leukemia ◽

Acute Myeloid

Donor cell leukemia is postulated to account for up to 5% of all leukemia "relapses" after hematopoietic stem cell transplant (SCT), though in many cases this is the first leukemia diagnosis for the patient if their transplant was for non-leukemia primary diseases. The rarity of the condition and heterogeneity of disease create challenges in diagnosis and management. In the present case, donor cell leukemia (DCL) developed in a 68-year-old female after allogeneic SCT 18 years earlier for follicular lymphoma. Only one other case of DCL after transplantation for follicular lymphoma has been reported (Boulton-Jones et al., Bone Marrow Transplantation, 2005). Furthermore, this case is atypical in that the presentation occurred many years after transplantation, since very few cases of DCL occur more than 15 years after original transplant. Case In 1993, the patient was diagnosed with stage IIIA follicular lymphoma at age 50. She achieved a complete remission with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) for 4 years. She relapsed in 1998 and received treatment with fludarabine and mitoxantrone. In 1999, she enrolled in a toxitumomab clinical trial (NCT00268203) but discontinued therapy secondary to side effects. Due to persistent disease, she proceeded with SCT and received EPOCH-F (etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and fludarabine) prior to allogeneic SCT from her brother in 2000 (6/6 HLA match), augmented with TH2 cells. She received graft versus host disease (GVHD) prophylaxis with cyclosporine, however her post transplant course was complicated by engraftment syndrome and gastrointestinal and skin GVHD. In 2019, she presented to hematology for evaluation of worsening chronic neutropenia and thrombocytopenia persistent for three years, noted during work-up for symptomatic cholelithiasis. Bone marrow biopsy revealed acute myeloid leukemia (AML) with a hypocellular marrow with 30% blasts and myelodysplasia related changes. Her cytogenetics showed 46XY, +1, der(1;7)(q10;p10)/47,sl,+8/46,XY. FISH analyses demonstrated deletion 7q31 D7S486 locus in 156/200 cells (78%). NGS panel showed IDH1 (VAF16%) and U2AF1 (VAF 26%) mutations. Based on cytogenetics and chimerism studies showing 100% donor, the patient was diagnosed with donor-derived AML secondary to allogeneic SCT from her brother. The brother currently has no known hematologic problems. The patient was treated with CPX-351 (liposomal cytarabine and daunorubicin) and achieved a complete remission, followed by consolidation with CPX-351. Given her complex cytogenetics and poor prognosis, the patient proceeded to non-myeloablative haploidentical peripheral blood SCT from her son, with post-transplant cyclophosphamide. She subsequently had complications of neutropenic fever and C. dificile colitis, with progressive colitis leading to her death on day 22 after SCT. Discussion Though cytogenetic and molecular studies along with functional status assist clinicians in treatment decisions for DCL patients, the benefits and risks of treatment remain difficult to balance for this unique subset of leukemia. Of patients that achieve remission for greater than 18 months, many undergo second allogeneic SCT, however a similar number of patients have remissions of at least 18 months treated with chemotherapy alone (Wiseman, Biology of Blood and Bone Marrow Transplantation, 2011). In 15 reported cases that went to SCT, approximately 50% lived longer than 12 months after their DCL diagnosis. Second allogeneic SCT is often favored after initial remission in patients with good performance status due to high risk for relapse. This case illustrates the challenge in management of donor cell leukemia, a rather rare entity with very few cases in the literature developing greater than 15 years after transplant. Limited robust evidence favoring a particular treatment supports the need for further prospective studies. Disclosures Lai: Agios: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Speakers Bureau; Astellas: Speakers Bureau.

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Therapy-Related Myeloid Neoplasm Has a Distinct Pro-Inflammatory Bone Marrow Microenvironment and Delayed DNA Damage Repair

Blood ◽

10.1182/blood-2020-137277 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 37-38

Author(s):

Monika M Kutyna ◽

Li Yan A Wee ◽

Sharon Paton ◽

Dimitrios Cakouros ◽

Agnieszka Arthur ◽

...

Keyword(s):

Dna Damage ◽

Board Of Directors ◽

Research Funding ◽

High Dose ◽

Cytotoxic Therapy ◽

Advisory Committees ◽

Myeloid Neoplasm ◽

Myeloid Neoplasms ◽

Damage Repair

Introduction: Therapy-related myeloid neoplasms (t-MN) are associated with extremely poor clinical outcomes in otherwise long-term cancer survivors. t-MN accounts for ~20% of cases of myeloid neoplasms and is expected to rise due to the increased use of chemotherapy/radiotherapy (CT/RT) and improved cancer survivorship. Historically, t-MN was considered a direct consequence of DNA damage induced in normal hematopoietic stem cells (HSC) by DNA damaging cytotoxics. However, these studies have largely ignored the bone marrow (BM) microenvironment and the effects of age and concurrent/previous cancers. Aim: We performed an exhaustive functional study of mesenchymal stromal cells (MSC) obtained from a comparatively large cohort of t-MN patients and carefully selected control populations to evaluate the long-term damage induced by cytotoxic therapy to BM microenvironment and its impact on malignant and normal haematopoiesis. Methods: Four different cohorts were used: (1) t-MN, in which myeloid malignancy occurred after CT/RT for a previous cancer (n=18); (2) patients with multiple cancer and in which a myeloid neoplasm developed following an independent cancer which was not treated with CT/RT (MC-MN; n=10); (3) primary MN (p-MN; n=7) untreated and without any prior cancer or CT/RT; (4) age-matched controls (HC; n=17). Morphology, proliferation, cellular senescence, differentiation potential and γH2AX DNA damage response was performed. Stem/progenitor supportive capacity was assessed by co-culturing haematopoietic stem cells on MSC feeder-layer in long-term culture initiating assay (LTC-IC). Cytokine measurements were performed using 38-plex magnetic bead panel (Millipore) and RNA sequencing libraries were prepared with Illumina TruSeq Total RNA protocol for 150bp paired-end sequencing on a NextSeq500 instrument. Functional enrichment analysis was performed using EnrichR software. Results: MSC cultured from t-MN patients were significantly different from HC, p-MN and MC-MN MSC according to multiple parameters. They exhibited aberrant morphology consisting of large, rounded and less adhesive cells compared to typical spindle-shaped morphology observed with controls. MSC from myeloid neoplasm also showed impaired proliferation, senescence, osteo- and adipogenic differentiation with t-MN MSC showing the greatest differences. DNA repair was dramatically impaired compared to p-MN and HC (Fig.1A). Importantly, these aberrant t-MN MSC were not able to support normal or autologous in vitro long-term haematopoiesis (Fig.1B). The biological characteristic and poor haematopoietic supportive capacity of MSC could be "cell-intrinsic" or driven by an altered paracrine inflammatory microenvironment. Interestingly, several inflammatory cytokines were higher in t-MN compared with marrow interstitial fluid obtained from p-MN patients (Fig.1Ci) and many of these including Fractalkine, IFNα2, IL-7 and G-CSF were also significantly higher in t-MN MSC conditional media (Fig.1Cii). Together, this data suggest that t-MN microenvironment is distinct from p-MN with paracrine production of pro-inflammatory milieu that may contribute to poor HSC supportive capacity. Preliminary whole transcriptome analysis revealed differential gene expression between t-MN and HC (Fig.1Di) and p-MN MSC. Importantly, the deregulated genes play critical role in cell cycle, DNA damage repair, and cellular senescence pathways explaining phenotypical characteristic of t-MN MSC (Fig.1Dii). Moreover CXCL12 expression, a key regulator of haematopoiesis, was significantly lower in t-MN compared to HC (p=0.002) and p-MN MSC (p=0.009), thus explaining poor HSC supportive capacity. The key difference between the p-MN, MC-MN and t-MN is prior exposure to CT/RT. To study this we obtained MSC from two t-MN patients for whom we had samples at the time of their primary cancer, post high-dose chemotherapy and at the time of t-MN. MSC displayed aberrant proliferation and differentiation capacity after high-dose cytotoxic therapy (2 to 4 years prior to developing t-MN) and remained aberrant at t-MN diagnosis (Fig.1E). Conclusions: BM-MSC from t-MN patients are significantly abnormal compared with age-matched controls and typical myeloid neoplasm. Importantly, prior CT/RT leads to long-term irreversible damage to the BM microenvironment which potentially contributes to t-MN pathogenesis. Disclosures Hughes: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Hiwase:Novartis Australia: Research Funding.

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The Metabolomic Status of the Differentiating Myeloid Lineage in MDS with Low and High Bone Marrow Blast Counts

Blood ◽

10.1182/blood-2020-140468 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 32-33

Author(s):

Aikaterini Poulaki ◽

Theodora Katsila ◽

Ioanna E Stergiou ◽

Stavroula Giannouli ◽

Jose Carlos Gόmez Tamayo ◽

...

Keyword(s):

Bone Marrow ◽

Board Of Directors ◽

Warburg Effect ◽

Research Funding ◽

Metabolic Reprogramming ◽

Negative Ion ◽

Advisory Committees ◽

Myeloid Lineage ◽

Epigenetic Modifier ◽

Cellular Fate

Despite its major role in cellular biology, metabolism has only recently acquired a principal role in the research of the most profound cellular cycle disturbance, cancerous transformation. Myelodysplastic syndromes (MDS), a massively heterogeneous group of Hematopoietic Stem/ Progenitor Cell (HSC/HPC) disorders lie at the interface of normal differentiation and malignant transformation and have thus drew great attention due to their polymorphic presentation and elusive pathophysiology. Failure to establish a direct etiopathogenic relationship with specific genetic aberrations, along with the novel finding of a highly deregulated HIF1 activity by several unrelated research groups worldwide, including ours, urged us to investigate the metabolomic status of human bone marrow derived differentiating myeloid lineage in comparison with one another as well as with control samples. BM aspiration samples collected from 14 previously untreated MDS patients (10 patients with <5% (1 SLD, 8MLD, 1del5q, group 1- G1) and 4 with >5% BM blasts (2 EB1, 2 EB2group 2 - G2)) and 5 age matched controls. Myeloid lineage cells were isolated through ficoll bilayer protocol. All samples contained homogenous myeloid lineage subpopulations, assessedthrough optical microscopy. Two different metabolite extraction protocols were applied. The one with the best metabolites yield (50% MeOH, 30% ACN, 20% H2O) was chosen. LC-MS/MS analysis was performed using UPLC 1290 system (Agilent Technologies) coupled to a TripleTOF 5600+ mass spectrometer (SCIEX) equipped with SWATH acquisition, SelexION technology and an electrospray ionization source (ESI). A threshold of a minimum of three samples expressing a given metabolite was set against data sparsity. Data tables were scaled by data centering and setting unit variance. Log2 Foldcalculation and PLS analysis were performed for the two datasets (positive and negative ion-modes). R2 and Q2 for positive ion-mode and negative-ion mode analyses were determined. Both datasets were merged in a unique data table by taking into account maximum absolute log2 foldvalues, when a metabolite was found in both datasets. Warburg effect was evidently present in both the G1 and G2 vs control comparisons, yet the role of this stem like aerobic glycolysis seems markedly different in the two groups. While in the G2 group it serves to rescue glucose from complete burn in the mitochondrion and thus shuts it towards nucleotide synthesis (Pentose Phosphate Pathway found upregulated) with the added benefit of increased reduced Glutathione synthesis and improved redox state, in the G1 group proves detrimental. This greatly variable effect of the same phenomenon in the cellular fate lies upon the quality and functionality of the cellular mitochondrial content. G2 precursors presented functional mitochondrial (decreased NAD/NADH and FAD/FADH2) contrary to the G1 ones (Table). Failing TCA cycle, with increased NAD/NADH and FAD/FADH2 ratios and markedly increased ADP/ATP levels leads to FAs accumulation due to failure of effective adequate β oxidation. The uncontrolled increase in the NAD/NADH ratio stimulates upper glycolysis into a turbo mode further increasing the ADP/ATP, depleting cellular energy contents, engaging it to a never-ending deadly metabolism. The enormous abundance of upper glycolytic intermediates is relieved through phospholipid and ceramide synthesis, all found massively upregulated in both the MDS vs control yet also in the G1 vs G2 comparisons. FAs, mostly phospholipid and ceramide accumulation, interrupt the mitochondrial membrane lipidome further incapacitating metabolic integrity and inducing their autophagic degradation which further stimulates the Warburg effect. This type of metabolic reprogramming is eventually targeted to epigenetic modifier production, increased S-adenosyl-methionine, the major methyl group donor, 2-HydroxyGlutarate, a potent epigenetic modifier and notorious oncometabolite, Acetyl-Lysine, the major acetyl- group donor, even glutathione. We therefore present a model of an uncontrolled Warburg effect which in the G1 group confers premature death of the hematopoietic precursors, the ineffective hematopoiesis of MDS. Yet, under the pressure of the vastly upregulated epigenetic modifiers cellular fate changes, the G1 precursors adapt and transform to the G2 ones yet eventually to Acute Myeloid Leukemia blasts. Table Disclosures Vassilopoulos: Genesis pharma SA: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

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