Germline Variants of RUNX-1 in Myeloid Malignancy

Abstract Mutation of the master transcription factor RUNX1 (NM_001754) has a well characterized role in the pathogenesis of myeloid neoplasms. RUNX1 is located at 21q22 and is composed of a c-terminal transactivation domain (TAD) (269-480) and an n-terminal RUNT domain (amino acid 76-209) that binds DNA and mediates heterodimerization with CBF-β. If the RUNT domain is disrupted a RUNX1 mutant will behave in a dominant negative fashion. Mutations leaving the RUNT domain intact result in haploinsufficiency compounded by CBF-β binding site competition with the wild-type allele. Germline (GL) RUNX1 mutations cause the autosomal dominant predisposition syndrome familial platelet disorder with propensity to myeloid malignancy (FPDMM). In FPDMM, RUNX1 mutations, including RUNT domain missense mutations or nonsense mutations throughout the gene, confer mild chronic platelet disorders and a lifetime risk of MDS/AML. As with other GL predisposition syndromes a secondary event is considered necessary for progression, typically an additional mutation in the wild type RUNX1 allele. Onset of MDS/AML occurs at a median age of 33 years but the latency can be variable and ease of detection in younger cases likely distorts summary statistics. Inherited mutations in adults may be difficult to distinguish and have not been systematically explored. We hypothesize that a fraction of patients with otherwise typical RUNX1-positive MDS or related disorders are in fact carriers of a RUNX1 GL mutation. DNA obtained from a cohort of 1451 patients with myeloid neoplasia was analyzed using a multiamplicon deep next-generation sequencing (NGS) panel including all ORFs of RUNX1. A total of 124 patients (8.5%) were found to carry 117 unique RUNX1 mutations. Of these, 59 hits were missense and 58 nonsense; and in 94 (76%) of cases the RUNT domain was disrupted. Eighteen (15.3% RUNX1 positive patients) were previously described in FPDMM. We applied various bio analytic criteria to designate somatic status in 57 patients. The remaining 64 RUNX1 mutations were further investigated, when possible, by paired capillary sequencing of CD3- mononuclear DNA and in-vitro expanded CD3+ T-Cell DNA. We found 5/31 (16%) of these cases carried mutation in both myeloid and lymphoid lineages and were designated GL. This included 2 post-RUNT truncations (p.Y281*, p.S410*) and two RUNT domain missense mutations (p.R80C, p.S141L) all of which are expected to produce a dominant negative phenotype. A 5th GL mutation of uncertain significance in the TAD (p.M310I) was also discovered. Excluding this TAD missense mutation, each of these have been described as somatic factors in myeloid disease. The truncation at amino acid 410 is further downstream than all reported FPDMM variants to date. Chart review revealed an anamnestic presence of thrombocytopenia in 80% of proposed FPD patients. Suggestive family history was found in both truncated cases, with hematologic malignancy presenting in a 1st degree relative before 30 years of age. Median age at diagnosis of MDS/AML in GL cases was 46 years (range: 18-68 years) compared to 65 years in the cases designated somatic (range: 37-87 years). Two cases (p.S410* and p.M310I) had cytogenetic abnormalities on the 21st chromosome (trisomy 21 and t(8;21) respectively) and had no other molecular abnormalities detected by our NGS panel. A mutation in GPR98 of unknown significance was found with p.Y281*. Lastly the p.S141L mutant had cooperating mutations in chromatin modifiers ASXL1 and BCOR, and in RNA splicing gene LUC7L2. This study of an exemplary GL leukemia gene suggests that systematic searches for known and potential GL predisposition genes in otherwise typical adult cohorts may reveal a GL role in the evolution of myeloid neoplasms. Future work to determine genetic predisposition to leukemia in adults is essential. Disclosures Sekeres: Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium/Takeda: Membership on an entity's Board of Directors or advisory committees. Carraway:Celgene: Research Funding, Speakers Bureau; Baxalta: Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen: 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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Distinct Clinical and Biological Implications of Various DNTMT3A Mutations in Myeloid Neoplasms

Blood ◽

10.1182/blood.v128.22.2872.2872 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2872-2872

Author(s):

Suresh Kumar Balasubramanian ◽

Mai Ali ◽

Taha Bat ◽

Bhumika Patel ◽

Bartlomiej P Przychodzen ◽

...

Keyword(s):

Bone Marrow ◽

Board Of Directors ◽

Clinical Features ◽

Bone Marrow Failure ◽

Missense Mutations ◽

Negative Consequences ◽

Wild Type ◽

Advisory Committees ◽

Myeloid Neoplasms ◽

Significant Difference

Abstract DNMT3A, a member of the DNA methyltransferases family along with DNMT1 and DNMT3B, is located on chromosome 2p23. Recurrent somatic mutations in DNMT3A are typically heterozygous and found mostly in non-CBF AML, less frequently in MDS and MPN. DNMT3A mutations are reported with other common myeloid mutations including NPM1, FLT3 and IDH1/2. The most canonical DNMT3A mutations are missense alteration in the R882 codon, accounting for >60% of all DNMT3A mutations and they imply dominant negative consequences. Overall, DNMT3A mutations carry a poor prognosis compared to the AML or MDS with wild type (WT) DNMT3A, although data within different subgroups (e.g., incorporating cytogenetic profiles) are conflicting. We hypothesized that molecular consequence of R882 mutations will differ from those of other somatic alterations of DNMT3A and may also result in distinct clinical features and outcomes. To test this theory, we analyzed a cohort of 1174 patients with myeloid neoplasias including 32% AML, 33% MDS, 13% MDS/MPN, 6% MPN and 16% other bone marrow failure disorders. These cases were subjected to multiamplicon targeted deep NGS including all ORFs of DNMT3A and other recurrently mutated genes. After application of various bioanalytic algorithms, confirmatory sequencing and thus stringent exclusion of all artifacts and germline alterations, we identified 140 somatic mutant cases (12% of the cohort), including 89 missense mutations (53 at R882, 19 at R693 and 17 other non-canonical missense alterations) and 51 truncations/frame shifts (all heterozygous). There was an age-related increase in the incidence of DNMT3A mutations, with the peak occurrence at 35-40 yrs. of age. Mutations in DNMT3A were most common in AML (54% in primary (p) AML, 8% in secondary (s) AML) followed by MDS (28%), MDS/MPN (4%), MPN (3%) and other bone marrow failure disorders (3%). Mutation in the R693 codon and truncating mutations were most commonly associated with MDS (p=.013) and sAML (p=.0013) whereas mutation occurring in codon R882 and other non-canonical missense mutations were frequently associated with pAML (p=.00001). For the whole cohort, DNMT3A mutations were most frequently associated with NPM1 (21% vs 8%, p=.014), FLT3 (24% vs. 2%, p=.0001), and IDH1/2 (26% vs. 8%, p=.001), compared to wild type DNMT3A. However, PRC2 complex mutations were less likely to occur in the context of DNMT3A mutations (6% vs. 24%, p=.0006). Canonical R882 mutation was commonly associated with FLT3 (p=.03) mutations, while truncating mutations were not (p=.03). Analyses of clonal hierarchy by ranking of VAF values demonstrated that 53% of DNMT3A mutations were dominant (mean VAF 39%, range 5-93%) (n=74/140). When DNMT3A mutations were dominant, IDH 1/2 (14%), TET2 (9%), ASXL (5%), PRC2 complex (3%) and BCOR (3%) mutations were common secondary events. In subgroup analyses, 55% of mutations in the R693 codon were dominant compared to 45% in R882 and 47% in truncating mutations. TET2 mutations were the most common associated secondary hits in dominant R693 mutations (n=10) compared to truncating (n=24) and R882 mutations (n=23) (40% vs. 8% vs. none, p=.0001). When DNMT3A mutations are secondary (mean VAF 34%, range 1-60%), as in 47% of our cases (n=66/140), then the common first hits were TET2 (10%), U2AF1 (8%) and cohesin complex (RAD21, SMC3, STAG2) mutations (6%). Dominant DNMT3A mutations correlated with MDS/MPN (60%, p=.007), while secondary DNMT3A mutations correlated with sAML (73%, p=.001). DNMT3A mutant myeloid neoplasms showed worse survival (p<.0001) compared to WT cases. Among different subgroups, there was significant difference in OS between R882, R693, truncating and other non-canonical missense mutations (p=.013). The R882 mutations had worse survival compared to other DNMT3A mutations (p=.003). Non-canonical mutations (truncating and other missense) vs. canonical mutations (R882 and R693) had better survival (p<.04). Survival for mutant R882 DNMT3A was worse compared to truncating mutations (p=.005) while there was no difference between R693 and truncating mutations. Among AML cases, R882 mutations vs. other mutations had worse survival (p=.01) while in MDS and MDS/MPN there was no significant difference in OS. DNTMT3A mutations often occur as founder lesion in AML. Our study shows that different types of mutations other than canonical R882 alterations may have a differential impact on OS and distinct clinical features. Disclosures Carraway: Celgene Corporation: Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees; Baxalta: Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees.

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CCND1 and CCND2 Mutations Are Frequent in Adults with Core-Binding Factor Acute Myeloid Leukemia (CBF-AML) with t(8;21)(q22;q22)

Blood ◽

10.1182/blood.v128.22.2740.2740 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2740-2740

Author(s):

Ann-Kathrin Eisfeld ◽

Jessica Kohlschmidt ◽

Sebastian Schwind ◽

Deedra Nicolet ◽

James S. Blachly ◽

...

Keyword(s):

Cell Cycle ◽

Amino Acid ◽

Board Of Directors ◽

Sanger Sequencing ◽

Small Sample ◽

Wild Type ◽

Advisory Committees ◽

Cell Counts ◽

Recurrent Mutations ◽

Frequent Mutations

Abstract CBF-AML is defined by the presence of either t(8;21)(q22;q22)/RUNX1-RUNX1T1 or inv(16)(p13.1q22)/t(16;16)(p13.1;q22)/CBFB-MYH11. The resulting fusion genes require a "second hit" to initiate leukemogenesis. Although compared with other AML subtypes patients with CBF-AML have a relatively favorable prognosis, still almost 40% of CBF-AML patients experience relapses of their disease. Mutation assessment of 84 leukemia- and/or cancer-associated genes using a customized targeted next-generation sequencing (NGS) approach was done on samples from 177 adults with CBF-AML [t(8;21), n=68; inv(16)/t(16;16), n=109]. We identified frequent mutations in the CCND1 and CCND2 genes as novel molecular alterations in AML with t(8;21). They were detected in 10 (15%) patients with t(8;21), making CCND1/2 mutations the third most frequently detected mutations in t(8;21) AML patients. In contrast, we found a single CCND2 mutation in only 1 (0.9%) patient with inv(16), and CCND1/2 mutations in only 11 of 1,426 non-CBF-AML patients (0.77%). Testing for CCND1 and CCND2 mutations using Sanger sequencing in additional 25 CBF-AML patients with t(8;21), for whom NGS analysis was not possible, identified 3 CCND1/2 mutated patients, thereby confirming the CCND1/2 mutation frequency in t(8;21) AML (15% in the NGS set, and 12% in Sanger sequencing set). All CCND2 mutations cluster around the highly conserved amino acid residue threonine 280 (Thr280), and encode a degradation-resistant CCND2 protein. Curiously, de novo germline CCND2 mutations affecting the identical amino acid residues Thr280 or Pro281 have been recently found to cause megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome (MPPH) type 3, an extremely rare neurodegenerative disorder (Mirzaa et al., Nat Genet. 2014;46:510-5). The variant allele fractions of the detected CCND1/2 mutations were variable, suggesting that these mutations can represent both early and later mutational events. Interestingly, three of the 11 CCND1/2 mutated patients had two different CCND1/2 mutations, suggesting a dependence of their leukemia on this mutational event. The evaluation of mutations in other genes found in CBF-AML patients with CCND1/2 mutations did not reveal any statistically significant co-occurring or mutually exclusive mutations. The most frequent mutation co-occurring with CCND1/2 was ASXL2, detected in 4 of the 10 (40%) cases with t(8;21). Patients with CCND1/2 mutations presented with lower percentages of blood (P=0.02) and bone marrow (P=0.05) blasts than patients without these mutations, but did not differ significantly with regard to other pretreatment characteristics. No differences in the achievement of complete remission, disease-free or overall survival were observed between CCND1/2 mutated and CCND1/2 wild-type patients. Mechanistically, we show that Thr280Ala mutated CCND2 significantly increased cell growth (as assessed by TiterGlo assays and cell counts) in all experimental set-ups. Western blotting revealed an increase of retinoblastoma gene phosphorylation, indicating increased downstream signaling. Moreover, both propidium iodide mediated cell cycle analysis and immunofluorescence staining for phospho-histone H3 indicated an increased mitotic rate and a decreased G1 fraction. In summary, recurrent mutations in CCND1/2 are consistent with the currently known mutational landscape of t(8;21) AML, which is dominated by receptor tyrosine kinase, RAS, and chromatin remodeling gene mutations─all of which can result in targeting of CCND1/2. While CCND1 and CCND2 play a crucial role in the regulation of hematopoietic differentiation and cell proliferation, and their differential activation has been implicated in leukemogenesis, frequent mutations in these genes have not previously been reported in AML. The fact that no outcome difference was seen when comparing CCND1/2-mutated versus wild-type patients may be due to the relatively small sample size, or may possibly reflect the common use of cell cycle affecting high-dose cytarabine-based therapy in CBF-AML patients. Thus, the identification of CCND1/2 mutations as frequent mutational events in t(8;21) AML may provide further justification for cell cycle-directed therapy in this disease. Disclosures Stone: Merck: Consultancy; Karyopharm: Consultancy; Jansen: Consultancy; Pfizer: Consultancy; Juno Therapeutics: Consultancy; ONO: Consultancy; Roche: Consultancy; Seattle Genetics: Consultancy; Sunesis Pharmaceuticals: Consultancy; Xenetic Biosciences: Consultancy; Celator: Consultancy; Agios: Consultancy; Amgen: Consultancy; Novartis: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees.

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Clinical and Biological Implications of CUX1 Mutations in Myeloid Neoplasms

Blood ◽

10.1182/blood.v128.22.3156.3156 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3156-3156

Author(s):

Mai Aly ◽

Naoko Hosono ◽

Przychodzen Bartlomiej ◽

Hideki Makishima ◽

Nagata Yasunobu ◽

...

Keyword(s):

Board Of Directors ◽

Lower Risk ◽

Somatic Mutations ◽

Myeloproliferative Neoplasms ◽

Loss Of Function ◽

Wild Type ◽

Advisory Committees ◽

Molecular Features ◽

Myeloid Neoplasms ◽

Normal Cytogenetics

Abstract Recurrent somatic mutations of CUX1 are described in myeloid neoplasms. CUX1 is located at chromosome 7q22.1; -7/del(7q) involving CUX1 locus are common abnormalities in myelodysplastic syndromes (MDS). Mutations and loss of heterozygosity involving CUX1 have been also described in breast, lung and uterine cancers. Preliminary functional studies, lack of a mutational hotspot and coincidental deletions suggest loss of function/hypomorphic consequences of these molecular defects. CUX1 (p200), contains 4 evolutionarily conserved DNA-binding domains, including 3 CUT repeats and a CUT homeodomain. Functionally, CUX1 regulates many genes involved in DNA replication and chromosome segregation. Cell-based assays have established a role for CUX1 in the control of cell-cycle progression, cell motility, and invasion .The objective of this study is to assess the molecular context and clinical significance of CUX1 mutations and deletions in myeloid neoplasms. We analyzed a subset of 1478 patients [24% lower-risk MDS, 17% higher-risk MDS, 22% primary (p)AML, 14% secondary AML, 14% MDS/myeloproliferative neoplasms (MPN) and 9% MPN] for the presence of CUX1 mutations and deletions. No CUX1 mutations were found in core binding factor AML. We correlated the presence of these lesions with clinical parameters, cytogenetic abnormalities, and molecular features including clonal architecture and associated somatic mutations. Copy number variation and their boundaries were analyzed by Single Nucleotide Polymorphism (SNP) arrays and mutations by multiamplicon deep sequencing utilizing a panel targeting 60 most commonly mutated genes in myeloid neoplasms. In total cohort 4 % of patients had CUX1 mutations and 6% had locus deletions (affecting ch 7q commonly deleted region: 7q22.1) including 90% of del (7q) cases. Expression of CUX1 is significantly lower in AML with -7/del(7q) compared to AML with normal cytogenetics (p<.00001) and also in MDS with -7/del(7q) compared tohealthy controls (p=.004). Additionally, decreased expression of CUX1 was found in 15% of MDS and 8% of AML patients without -7/del(7q) or related mutations. Cases with lower expression had worse OS compared to patients with higher expression (p=.002). In terms of configuration, most mutations were heterozygous, 5% of mutations were hemizygous and 4% were homozygous (due to UPD). Among 75 somatic CUX1mutations; 72% were missense, 20% where frame shift and 8% where non sense. CUX1 mutations were associated with either lower-risk MDS (p=.0001) and pAML (p=.04) while deletions involving the CUX1 locus were significantly related to higher-risk MDS (p=.05). Heterozygous CUX1 mutations were more commonly associated with normal cytogenetics (p=.01). Patients with -7/del(7q) frequently represented del(5q) (p=.04) and thrombocytopenia (p=.001). The OS of patients with CUX1 mutations was shorter (p=.04) as was that of patients with CUX1/deletions (p=.02) when compared to wild type. We subsequently studied the molecular background of CUX1 alterations. CUX1 mutations (vs. wild type) were associated with TET2 (31% vs. 14%, p=.006), ASXL1 (29% vs. 9%, p=.0005), BCOR (28% vs. 8%, p=.0004), and cohesion mutations (26%, vs. 5%, p=.0005), while NPM1 mutations showed the reverse relationship (1% vs. 7%, p=.03). RAS and CUX1 mutations were mutually exclusive (0% vs. 6%, p=.03). When we analyzed clonal hierarchy in the context of CUX1 mutations; dominant CUX1 mutations (24%; mean VAF=49%); were accomplished by ASXL1 (21%) and SRSF2 (14%) mutations which were the most common secondary events in this context. Phenotypically, dominant CUX1 mutations were associated with MDS/MPN (42%) and MDS (33%). 14% of CUX1 mutant cases did not harbor any other alterations and were not associated with a discernable phenotype. Secondary CUX1 lesions (62%; mean VAF=22%) were found in the context of dominant TET2 mutations (16%). The pathomorphologic context of secondary CUX1 mutation did not differ from that of primary lesions. AML seemed to be underrepresented (p=.006) and MPN overrepresented (p=.019) among dominant CUX1 mutant cases. In conclusion, CUX1 lesions including locus deletions with haploinsuffciency, mutations and a fraction of cases with decreased CUX1 expression can be encountered in MDS and related neoplasms, chiefly AML. CUX1 dysfunction is associated with poor survival likely due to its distinct molecular background. Disclosures Makishima: The Yasuda Medical Foundation: Research Funding. Sekeres:Millenium/Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

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Mutations of the AML1 gene in myelodysplastic syndrome and their functional implications in leukemogenesis

Blood ◽

10.1182/blood.v96.9.3154 ◽

2000 ◽

Vol 96 (9) ◽

pp. 3154-3160 ◽

Cited By ~ 123

Author(s):

Yoichi Imai ◽

Mineo Kurokawa ◽

Koji Izutsu ◽

Akira Hangaishi ◽

Kengo Takeuchi ◽

...

Keyword(s):

Amino Acid ◽

Dna Binding ◽

Myelodysplastic Syndrome ◽

Binding Protein ◽

Single Letter ◽

Dominant Negative ◽

Wild Type ◽

Runt Domain ◽

Dominant Negative Inhibitor ◽

Aml1 Gene

Abstract The AML1 gene encodes a DNA-binding protein that contains the runt domain and is the most frequent target of translocations associated with human leukemias. Here, point mutations of the AML1 gene, V105ter (single-letter amino acid code) and R139G, (single-letter amino acid codes) were identified in 2 cases of myelodysplastic syndrome (MDS) by means of the reverse transcriptase–polymerase chain reaction single-strand conformation polymorphism method. Both mutations are present in the region encoding the runt domain of AML1 and cause loss of the DNA-binding ability of the resultant products. Of these mutants, V105ter has also lost the ability to heterodimerize with polyomavirus enhancer binding protein 2/core binding factor β (PEBP2β/CBFβ). On the other hand, the R139G mutant acts as a dominant negative inhibitor by competing with wild-type AML1 for interaction with PEBP2β/CBFβ. This study is the first report that describes mutations of AML1 in patients with MDS and the mechanism whereby the mutant acts as a dominant negative inhibitor of wild-type AML1.

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Mutations of the AML1 gene in myelodysplastic syndrome and their functional implications in leukemogenesis

Blood ◽

10.1182/blood.v96.9.3154.h8003154_3154_3160 ◽

2000 ◽

Vol 96 (9) ◽

pp. 3154-3160 ◽

Cited By ~ 6

Author(s):

Yoichi Imai ◽

Mineo Kurokawa ◽

Koji Izutsu ◽

Akira Hangaishi ◽

Kengo Takeuchi ◽

...

Keyword(s):

Amino Acid ◽

Dna Binding ◽

Myelodysplastic Syndrome ◽

Binding Protein ◽

Single Letter ◽

Dominant Negative ◽

Wild Type ◽

Runt Domain ◽

Dominant Negative Inhibitor ◽

Aml1 Gene

The AML1 gene encodes a DNA-binding protein that contains the runt domain and is the most frequent target of translocations associated with human leukemias. Here, point mutations of the AML1 gene, V105ter (single-letter amino acid code) and R139G, (single-letter amino acid codes) were identified in 2 cases of myelodysplastic syndrome (MDS) by means of the reverse transcriptase–polymerase chain reaction single-strand conformation polymorphism method. Both mutations are present in the region encoding the runt domain of AML1 and cause loss of the DNA-binding ability of the resultant products. Of these mutants, V105ter has also lost the ability to heterodimerize with polyomavirus enhancer binding protein 2/core binding factor β (PEBP2β/CBFβ). On the other hand, the R139G mutant acts as a dominant negative inhibitor by competing with wild-type AML1 for interaction with PEBP2β/CBFβ. This study is the first report that describes mutations of AML1 in patients with MDS and the mechanism whereby the mutant acts as a dominant negative inhibitor of wild-type AML1.

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Homoharringtonine and Mebendazole Are Preferentially Lethal Against Models of Myeloid Malignancy Associated with Germline Mutant RUNX1

Blood ◽

10.1182/blood-2021-152871 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1177-1177

Author(s):

Christopher Peter Mill ◽

Warren C. Fiskus ◽

Courtney D. DiNardo ◽

Christine Birdwell ◽

Arnold Salazar ◽

...

Keyword(s):

Protein Expression ◽

Cell Line ◽

Board Of Directors ◽

Research Funding ◽

Unmet Need ◽

Protein Synthesis Inhibitor ◽

Advisory Committees ◽

Myeloid Malignancy ◽

Nsg Mice ◽

Platelet Disorder

Abstract RUNX1 is a master-transcriptional regulator involved in normal and malignant hematopoiesis. Majority of mono-allelic germline mutations in RUNX1 are missense, large deletions or truncation mutations, behaving mostly as loss of function (LOF) mutations. They are ~40%-penetrant and cause Familial Platelet Disorder (RUNX1-FPD) that has a propensity to evolve into myeloid malignancy (FPD-MM), i.e., MDS or AML. FPD-MM harbors co-mutations, most commonly on the second allele of RUNX1, and on BCOR, PHF6, K-RAS, WT1 or TET2, which confer relative resistance to standard therapy for MDS or AML. Although curative in some patients with FPD-MM, allogeneic transplantation from matched, un-related donors carries risk of graft versus host disease and frequent AML relapse. This creates a strong rationale and an unmet need to develop novel targeted therapies for FPD-MM. We previously reported on utilizing the RNA-Seq signature of RUNX1 knockdown, which exerted more lethality in AML cells with mutant (mt) RUNX1 compared to AML harboring two copies of wild-type RUNX1, for conducting LINCS1000-CMap analysis. This identified several expression mimickers (EMs), including the protein synthesis inhibitor homoharringtonine (HHT or omacetaxine) and anthelmintic fenbendazole (analog of mebendazole). Present studies demonstrate that treatment with HHT or mebendazole (MB) dose-dependently induced significantly greater loss of viability in four patient-derived (PD) bone marrow aspirate (BMA) samples of FPD-MM (3 AML and 1 MDS) compared to RUNX1-FPD (3 samples) or in normal CD34+ progenitor cells. In a patient with RUNX1-FPD (expressing mtRUNX1 K194N), who developed FPD-MM, following co-mutations were documented by NGS: BCOR A1437fs, PHF6 L324fs, SF3B1 D781G and SRSF2 P95R/L. From BMA of this patient, we successfully established the first ever, continuously cultured cell line (GMR-AML1) expressing the same germline mtRUNX1. GMR-AML1 cells were cytogenetically diploid and lacked MYC or MLL1 rearrangement, or any copy number gains or losses on array CGH. However whole exome sequencing (WES) identified additional mutations in TP53 (P72R), AIM2 (K340fs), NELFB (L523F), CEP152 (Y370X), SUGP2 (H23L), RRM2B (R71fs), TADA3 (T27R), SPDYE6 (G292C) and PRDM9 (S814R) with % VAF ranging between 33 to 55%. GMR-AML1 cells exhibited high surface expression of CD117 (c-KIT), CD123 (IL3R), CD86 and CD33, but without expression of CD34, CD14, CD11b, MPO or CD135 (FLT3). Compared to the AML OCI-AML5 cells with somatic mtRUNX1, GMR-AML1 cells demonstrated markedly reduced protein expression of RUNX1, RUNX2, PU.1, c-Myb, GFI1, GFI1B, FLT3, MEIS1 and CEBPα (p42), but much higher protein expression of RUNX3 and NOTCH (p120). CRISPR-Cas9 knockout of RUNX3 in GMR-AML1 cells restored RUNX1 expression, while significantly increasing % of differentiated cells. Although dose-dependently sensitive to daunorubicin, etoposide, cytarabine and panobinostat (class I and II HDAC inhibitor), GMR-AML1 cells were relatively insensitive to venetoclax, A1155463 (Bcl-xL inhibitor), AZD-5991 (MCL1 inhibitor), azacytidine or decitabine. Notably, treatment with HHT or MB dose-dependently induced loss of viability of GMR-AML1 cells (LD50: 40 and 330 nM, respectively). Additionally, co-treatment with HHT and venetoclax synergistically induced apoptosis in GMR-AML1 cells, as determined by the SynergyFinder algorithm. This synergy in GMR-AML1 cells was associated with abrogation of venetoclax-induced increase in MCL1 and Bcl-xL levels, as well as greater decline in levels of RUNX3, PU.1, c-Myb, c-Myc, MPL and CDK4/6. Tail vein infusion and engraftment of luciferase-transduced GMR-AML1 (10 6 cells) caused marked splenomegaly and 100% mortality of NSG mice by day-18, post-infusion. We will present at the ASH meeting findings of ongoing in vivo studies determining effects of treatment with HHT and/or venetoclax, versus vehicle control, on AML burden and overall survival of NSG mice engrafted with GMR-AML1 cells. Overall, preclinical findings presented here highlight the molecular and genetic features associated with progression of RUNX1-FPD to FPD-MM, especially in the newly established GMR-AML1 cell line. They also demonstrate that HHT or MB are preferentially more lethal against FPD-MM versus RUNX1-FPD cells and exert synergistic lethality with venetoclax against GMR-AML1 cells. Disclosures DiNardo: GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; AbbVie: Consultancy, Research Funding; Agios/Servier: Consultancy, Honoraria, Research Funding; Notable Labs: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Research Funding; Takeda: Honoraria; ImmuneOnc: Honoraria, Research Funding; Forma: Honoraria, Research Funding; Foghorn: Honoraria, Research Funding; Celgene, a Bristol Myers Squibb company: Honoraria, Research Funding. Takahashi: GSK: Consultancy; Celgene/BMS: Consultancy; Symbio Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy. Khoury: Stemline Therapeutics: Research Funding; Angle: Research Funding; Kiromic: Research Funding.

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UTX mutations in Myeloid Neoplasms

Blood ◽

10.1182/blood.v128.22.3148.3148 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3148-3148

Author(s):

Yasunobu Nagata ◽

Tomas Radivoyevitch ◽

Hideki Makishima ◽

Cassandra M. Hirsch ◽

Bartlomiej P Przychodzen ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

De Novo ◽

Missense Mutations ◽

Loss Of Function ◽

Advisory Committees ◽

Female Ratio ◽

Myeloid Neoplasms ◽

De Novo Aml

Abstract Genetic studies in myeloid neoplasms have revealed that somatic mutations and deletions frequently affect epigenetic regulation via DNA methylation and histone modification. One frequently affected epigenetic complex is polycomb repressive complex 2 (PRC2), which trimethylate Histone3Lysine27 (H3K27) to create a repression mark for the expression of a variety of genes that includes essential homeobox genes. Members of this complex include EZH2, EED and SUZ12, which exert methyltransferase activity, and perhaps indirectly also DNTM3A and ASXL1. The histone demethylase ubiquitously transcribed X (UTX) counters the enzymatic activity of PRC2 by removing di- and trimethyl groups from H3K27. It thus removes repressive marks. We were the first to report UTX mutations in a small portion of CMML and MDS cases. In another cohort, frequent somatic loss-of-function mutations in UTX were found in ALL. The goal of the present study was to understand the functional role of UTX and the consequences of its mutations on H3K27 methylation status, specifically with respect to their relevance to myeloid neoplasia. A total of 1,979 patients with various myeloid neoplasms (n = 1,686) and other hematologic disorders (n = 293) were screened for gene mutations in UTXand other reported driver genes relevant to myeloid neoplasms using whole exome sequencing and/or targeted deep-sequencing. Expression array analyses were performed on 200 samples. In addition, we also used sequencing and expression data from the de novo AML TCGA repository. UTXwas mutated in 55 (2.8%) cases out of 1,979 cases. The mutations were found in 2.5% of MDS, 3.1% de novo AML (including 8.3% CBF AML), 4.8% MDS/MPN, and 1.4% secondary AML (sAML). Of those, 77% were missense mutations and 23% nonsense, splice site and frameshift mutations, without an evident hot spot. The male-to-female ratio was 1.2 (55% vs. 45%). UTX gene expression was significantly higher in females than in males (p<.001). After gender adjustment and dichotomized using a threshold of expression of 2 standard deviations from the mean, low UTX expression levels were identified in 13/183 (7%) individuals. When we focused on clonal burden using variant allele frequencies (VAFs) to investigate clonal architecture within the tumor population, in 37 cases UTX constituted subclonal events and in 18 they were dominant. We then examined the molecular context of UTX lesions. Average mutation burden in patients with UTX mutations was higher than in WT UTX carriers (4 vs. 1.5, p<.001). UTX mutations co-occurred with other driver genetic mutations such as ASXL1, ZRSR2, CUX1, NF1. Surprisingly, EZH2 mutations are also enriched in UTX mutated cases although they function in the opposite direction of H3K27 trimethylation. Focusing on dominant clone and subclonal events in cases with these two mutations, EZH2mutations were enriched in cases with dominant UTX clone. This suggests that they play important roles in the cancer's pathogenesis. To clarify the role of UTX in the maintenance of leukemia, genomic knockouts of UTX were developed in human leukemic cell lines using the CRISPR-Cas9 system. RNA sequencing revealed that knockout cell enrichment for developmental regulators such as Hox genes. In addition, we made knockdowns of human CD34+ cells using short hairpin RNAs against UTX. The cells showed enhanced colony formation and increased replating efficiency consistent with retained clonogenicity. The truncating pattern of UTX mutations in myeloid neoplasms suggests that they are loss-of-function hits. Missense mutations thus need to be confirmed. Functional analysis in vitro shows that low expression of UTX may have functionally equivalent consequences. If so, an additional 7% of patients may have low UTX expression, and may thus phenocopy patients with UTX mutations. Combined, a total of ~10% out of myeloid neoplasm patients may harbor UTX deficiency. Epigenetic modifying drugs related to H3K27 such as inhibitors of EZH2and histone deacetylases are in development. It is controversial to which patients they should be applied. Our findings could be key to a deeper understanding of epigenetic alterations, drug function, and response. Disclosures Makishima: The Yasuda Medical Foundation: Research Funding. Mukherjee:Novartis: Consultancy, Honoraria, Research Funding; Ariad: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding. Carraway:Celgene: Research Funding, Speakers Bureau; Baxalta: Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Sekeres:Millenium/Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

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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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Antibody Response to Vaccination with BNT162b2, mRNA-1273, and ChADOx1 in Patients with Myeloid and Lymphoid Neoplasms

Blood ◽

10.1182/blood-2021-146832 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 218-218

Author(s):

Jil Rotterdam ◽

Margot Thiaucourt ◽

Juliana Schwaab ◽

Andreas Reiter ◽

Sebastian Kreil ◽

...

Keyword(s):

Antibody Response ◽

Board Of Directors ◽

Research Funding ◽

Low Grade ◽

Hematological Disease ◽

Advisory Committees ◽

Hematological Diseases ◽

Myeloid Neoplasms ◽

Lymphoid Neoplasms ◽

Treatment Naïve

Abstract Background: In general, patients with hematological diseases are predisposed to develop infections. Severe COVID-19 infection associated with high mortality is more likely in these patient cohorts compared to the general population. Due to immune defects related to the primary disease and/or to immunosuppressive treatment regimes, vaccination efficacy may be reduced in patients with hematological diseases. So far, data on this area are limited. Aim: To evaluate vaccination-related antibody response to BNT162b2, mRNA-1273, and ChADOx1 in patients with hematological disorders. Patients and methods: In this interim analysis of a prospective, observational single-center study, we report antibody levels at least 2 weeks after COVID-19 vaccination. A FDA/CE approved electrochemiluminescent assay (ECLIA) (Elecsys®, Roche, Mannheim, Germany) was used to quantify antibodies, pan Ig (including IgG) against the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. The assay has a measurement range of 0.4 to 250 U/mL, with a concentration ≥0.8 U/ml considered as positive. Data were analyzed for patients without detection of anti-N (nucleocapsid) SARS-CoV-2 antibody (i.e., without having passed SARS-CoV-2 infection). All tests were performed according to the manufacturer's instructions in an accredited laboratory at the University Hospital Mannheim. Results: Between February 2021 and July 2021, a total of 175 patients with hematological diseases were included in this study. The median age was 66 years (range 21-90 years), and 81 (46.3%) were female. The antibody levels were measured at least 14 days (median, 58 days) after the 2 nd vaccination. The patients were vaccinated with BNT162b2 (BioNTech, n=134), mRNA-1273 (Moderna, n=19), ChADOx1 (AstraZeneca, n=12), or got the first vaccination with BNT162b2 and the second with ChADOx1 (n=10). Overall, 145/175 (82.9%) were diagnosed with a malignant hematological disease (myeloid neoplasms, n=108; lymphoid neoplasms, n=37) and 30/175 with a non-malignant hematological disease (autoimmune disease, n=24; benign, n=6). 124 patients (70.1%) were on active therapy, and 51 patients (29.1%) were previously treated or treatment naïve. Correlation to specific therapies is ongoing and will be presented. In general, vaccination-related antibody response was positive (≥0.8 U/mL) in 148/175 (84.6%) patients with a median level of 208.6 U/mL (range 0.8-250.00) and negative (<0.8 U/mL) in 27/175 (15.4%) patients. The distribution of the negative cohort regarding the disease subgroups were as followed: myeloid neoplasms 7/27 (25.9%), lymphoid neoplasms 16/27 (59.3%), non-malignant hematological disease 4/27 (14.8%). Within the negative cohort, 21/27 (77.8%) were treated on active therapy, 6/27 (22.2%) were previously treated or treatment naïve. In myeloid neoplasms, patients with classical myeloproliferative neoplasm (MPN) had the highest negative result for antibodies with 4/7 (57.1%) followed by myelodysplastic syndrome (MDS) 2/7 (28.6%). Interestingly, all patients with chronic myeloid leukemia (CML) had a measurable immune response. In lymphoid neoplasms, patients with low-grade non-hodgkin lymphoma (NHL) (predominately chronic lymphocytic leukemia, CLL) had the highest negative antibody result 13/16 (81.3%) followed by high-grade NHL 4/8 (50%; predominately diffuse large b-cell lymphoma, DLBCL). In non-malignant hematological diseases, only patients with autoimmune diseases had a negative result. Conclusion: A remarkable group of patients with hematological disease were measured with no or low immune response after 2 nd COVID-vaccination, especially those with low-grade NHL, MDS and autoimmune disease. It seems that the percentage of patients with MPN and low response is less critical. No problems appeared in CML patients. Further explorations are needed with focus on potential risk of COVID infections despite full vaccination: The potential of 3 rd booster vaccination should be explored within clinical trials. Disclosures Reiter: AOP Orphan Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel support; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses, Research Funding; Celgene/BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel support; Incyte: Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses; Blueprint Medicines: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses; Abbvie: Membership on an entity's Board of Directors or advisory committees; Deciphera: Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses. Kreil: Novartis: Research Funding. Hofmann: Amgen: Honoraria; BMS: Honoraria; Novartis: Honoraria. Jawhar: Takeda: Honoraria, Other: Travel support; Blueprint Medicines: Honoraria; Stemline: Consultancy, Honoraria; Celgene: Other: Travel support; Novartis: Consultancy, Honoraria, Other: Travel support, Speakers Bureau. Saussele: Roche: Honoraria; Pfizer: Honoraria; Incyte: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

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A 12-Amino-Acid Segment, Present in Type s2 but Not Type s1 Helicobacter pylori VacA Proteins, Abolishes Cytotoxin Activity and Alters Membrane Channel Formation

Journal of Bacteriology ◽

10.1128/jb.183.22.6499-6508.2001 ◽

2001 ◽

Vol 183 (22) ◽

pp. 6499-6508 ◽

Cited By ~ 76

Author(s):

Mark S. McClain ◽

Ping Cao ◽

Hideki Iwamoto ◽

Arlene D. Vinion-Dubiel ◽

Gabor Szabo ◽

...

Keyword(s):

Helicobacter Pylori ◽

Amino Acid ◽

Dominant Negative ◽

Membrane Channels ◽

Wild Type ◽

Amino Terminal ◽

Protein Toxin ◽

H Pylori ◽

Amino Acid Segment ◽

Negative Phenotype

ABSTRACT Helicobacter pylori, a gram-negative bacterium associated with gastritis, peptic ulceration, and gastric adenocarcinoma in humans, secretes a protein toxin, VacA, that causes vacuolar degeneration of epithelial cells. Several different families of H. pylori vacA alleles can be distinguished based on sequence diversity in the “middle” region (i.e., m1 and m2) and in the 5′ end of the gene (i.e., s1 and s2). Type s2 VacA toxins contain a 12-amino-acid amino-terminal hydrophilic segment, which is absent from type s1 toxins. To examine the functional properties of VacA toxins containing this 12-amino-acid segment, we analyzed a wild-type s1/m1 VacA and a chimeric s2/m1 VacA protein. Purified s1/m1 VacA from H. pylori strain 60190 induced vacuolation in HeLa and Vero cells, whereas the chimeric s2/m1 toxin (in which the s1 sequence of VacA from strain 60190 was replaced with the s2 sequence from strain Tx30a) lacked detectable cytotoxic activity. Type s1/m1 VacA from strain 60190 formed membrane channels in a planar lipid bilayer assay at a significantly higher rate than did s2/m1 VacA. However, membrane channels formed by type s1 VacA and type s2 VacA proteins exhibited similar anion selectivities (permeability ratio, PCl/PNa = 5). When an equimolar mixture of the chimeric s2/m1 toxin and the wild-type s1/m1 toxin was added to HeLa cells, the chimeric toxin completely inhibited the activity of the s1/m1 toxin. Thus, the s2/m1 toxin exhibited a dominant-negative phenotype similar to that of a previously described mutant toxin, VacA-(Δ6–27). Immunoprecipitation experiments indicated that both s2/m1 VacA and VacA-(Δ6–27) could physically interact with a c-myc epitope-tagged s1/m1 VacA, which suggests that the dominant-negative phenotype results from the formation of heterooligomeric VacA complexes with defective functional activity. Despite detectable differences in the channel-forming activities and cytotoxic properties of type s1 and type s2 VacA proteins, the conservation of type s2 sequences in many H. pyloriisolates suggests that type s2 VacA proteins retain an important biological activity.

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