5q Myelodysplastic Syndromes Research Articles

Low dose lenalidomide versus placebo in non-transfusion dependent patients with low risk, del(5q) myelodysplastic syndromes (SintraREV): a randomised, double-blind, phase 3 trial

Lenalidomide is the standard of care for patients who are transfusion dependent with chromosome 5q deletion (del[5q]) myelodysplastic syndromes. In the SintraREV trial, we aimed to investigate whether an early intervention of low lenalidomide doses for 2 years could delay transfusion dependency in patients with anaemia who were not transfusion dependent. This randomised, double-blind, phase 3 trial, was conducted at 22 sites (University Hospitals) in Spain, France, and Germany. Eligible patients were aged 18 years or older diagnosed with low-risk or intermediate-1-risk del(5q) myelodysplastic syndromes with non-transfusion-dependent anaemia (according to the IPSS), were erythropoietin-stimulating agents naive, and had an ECOG performance status of 2 or less. Patients were randomly assigned (2:1) by means of a telephone system to receive lenalidomide 5 mg daily in 28-day cycles versus placebo for 2 years. The primary endpoint was time to transfusion dependency based on blinded independent central review. Analysis were by intent-to-treat (ITT) and evaluable population. Safety analyses included all participants who received at least one dose of treatment. This trial is registered with ClinicalTrials.gov (NCT01243476) and EudraCT (2009-013619-36) and is complete. Between Feb 15, 2010, and Feb 21, 2018, 61 patients were randomly assigned to receive lenalidomide (n=40; two did not receive treatment) or placebo (n=21). The median age was 72·2 (IQR 65·4-81·9) years, 50 (82%) patients were female, and 11 (18%) were male. The median follow-up time was 60·6 (IQR 32·1-73·9) months. Regarding primary endpoint, median time to transfusion dependency was not reached (95% CI not applicable) in the lenalidomide group versus 11·6 months (95% CI 0·00-30·11) in the placebo group (p=0·0027). Lenalidomide significantly reduced the risk of transfusion dependency by 69·8% (hazard ratio 0·302, 95% CI 0·132-0·692; p=0·0046). The most frequent treatment-related adverse event was neutropenia, occurring in 24 (63%) of 38 patients in the lenalidomide group (grade 3 and 4 in 17 [45%] patients and one [3%], respectively) and in four (19%) of 21 patients in the placebo group (grade 3 in one [5%] patient). Thrombocytopenia was detected in seven (18%) of 38 patients receiving lenalidomide (grade 3 in two [5%] patients). Regarding the non-haematological toxicity, skin disorders (rash nine [23%] of 38 patients) were the most frequently described toxicities among patients receiving lenalidomide, being grade 3 in one (3%) of 38 patients. 19 serious adverse events were reported in 13 patients, 18 in the lenalidomide group and one in the placebo group, five of which were potentially related to the study drug. No treatment-related deaths were identified. An early approach with low doses of lenalidomide across two years delays the time to transfusion dependency and improves the rate and quality of the responses, with a manageable safety profile in patients who are non-transfusion dependent with del(5q) low-risk myelodysplastic syndromes. Bristol Myers Squibb.

Treatment characteristics and outcomes in lower-risk, non-del(5q) myelodysplastic syndromes: findings from a medical record review in the USA, Canada and Europe.

Aim: To assess treatment patterns and outcomes in patients with non-del(5q) lower-risk myelodysplastic syndromes.Methods: Patient medical records were reviewed in the USA, Canada (CAN), UK and the EU.Results: Analysis included 119 patients in the USA/CAN (median age, 61.5years) and 245 patients in the UK/EU (median age, 67.3years). Most patients received erythropoiesis-stimulating agents (ESAs) as first-line (1L) therapy (USA/CAN: 89.0%; UK/EU: 90.2%). A substantial proportion of 1L erythropoiesis-stimulating agent-treated patients were transfusion dependent before 1L (USA/CAN: 37.1%; UK/EU: 51.2%); a small percentage of these patients achieved transfusion independence during 1L therapy (USA/CAN: 2.8%; UK/EU: 14.4%).Conclusion: These findings highlight an unmet need for more effective treatments among patients with non-del(5q) lower-risk myelodysplastic syndromes.

Single-cell transcriptional profile of CD34+ hematopoietic progenitor cells from del(5q) myelodysplastic syndromes and impact of lenalidomide

While myelodysplastic syndromes with del(5q) (del(5q) MDS) comprises a well-defined hematological subgroup, the molecular basis underlying its origin remains unknown. Using single cell RNA-seq (scRNA-seq) on CD34+ progenitors from del(5q) MDS patients, we have identified cells harboring the deletion, characterizing the transcriptional impact of this genetic insult on disease pathogenesis and treatment response. Interestingly, both del(5q) and non-del(5q) cells present similar transcriptional lesions, indicating that all cells, and not only those harboring the deletion, may contribute to aberrant hematopoietic differentiation. However, gene regulatory network (GRN) analyses reveal a group of regulons showing aberrant activity that could trigger altered hematopoiesis exclusively in del(5q) cells, pointing to a more prominent role of these cells in disease phenotype. In del(5q) MDS patients achieving hematological response upon lenalidomide treatment, the drug reverts several transcriptional alterations in both del(5q) and non-del(5q) cells, but other lesions remain, which may be responsible for potential future relapses. Moreover, lack of hematological response is associated with the inability of lenalidomide to reverse transcriptional alterations. Collectively, this study reveals transcriptional alterations that could contribute to the pathogenesis and treatment response of del(5q) MDS.

Abstract 248: HSPA9/mortalin regulates erythroid maturation through a TP53-dependent mechanism in human CD34+ hematopoietic progenitor cells

Abstract Mortalin, encoded by the HSPA9 gene, is a highly conserved heat-shock chaperone belonging to the HSP70 family. It is predominantly presented in the mitochondria, and is critical in regulating a variety of cell physiological functions such as response to cell stress, control of cell proliferation, and inhibition/prevention of apoptosis. Myelodysplastic syndromes (MDS) are a group of hematopoietic stem cell malignancies characterized by ineffective hematopoiesis, increased apoptosis of bone marrow cells, and anemia. Up to 25% of MDS patients harbor an interstitial deletion on the long arm of chromosome 5, also known as del(5q), creating haploinsufficiency for multiple genes including HSPA9. Our prior study showed that knockdown of HSPA9 induces TP53-dependent apoptosis in human CD34+ hematopoietic progenitor cells, consistent with cytopenia observed in MDS patients. Since anemia is another featured symptom of MDS, we hypothesize that HSPA9 plays a role in regulating erythroid maturation. To test our hypothesis, we inhibited the expression of HSPA9 using various methods and measured the erythroid maturation in human CD34+ cells. We used siRNA targeting HSPA9 and found that HSPA9 siRNA significantly inhibited the cell growth, increased cell apoptosis, inhibited erythroid maturation (using CD71 as a surrogate marker), and increased p53 expression (p<0.01) compared to control siRNA in human CD34+ cells. Pharmacologic inhibition of HSPA9 by the chemical MKT-077, an inhibitor of HSP70 protein family members including mortalin, also increased p53 expression and inhibited erythroid maturation in human CD34+ cells. In addition, knockdown of HSPA9 by shRNA showed significant inhibition of erythroid maturation in human CD34+ cells compared to control shRNA. In order to test whether the regulation of erythroid maturation by HSPA9 is TP53-dependent or not, we constructed shRNAs targeting TP53 genes and simultaneously transduced lentivirus containing shRNAs targeting HSPA9 and TP53 respectively in human CD34+ cells using double antibiotics selection (puromycin for shRNA targeting HSPA9 and hygromycin for shRNA targeting TP53). We found that TP53 knockdown partially rescued the erythroid maturation defect caused by HSPA9 inhibition, suggesting that erythroid maturation inhibition by HSPA9 knockdown is partly mediated through a TP53 mechanism. Collectively, our results suggest that the increased apoptosis and reduced erythroid maturation observed in del(5a)-associated MDS is TP53-dependent. HSPA9/mortalin may be a potential target to treat anemia in del(5q) MDS patients, although simultaneous loss of multiple genes on del(5q) likely contributes to the complex phenotypes observed in MDS. Thus, our study not only uncovers some underlying mechanisms of del(5q) MDS, but also provides potential therapeutic indications through gene targeting in clinical MDS treatment. Citation Format: Tuoen Liu, Christopher Butler, Morgan Dunmire, Gabor Szalai, Anissa Johnson, Jaebok Choi, Matthew Walter. HSPA9/mortalin regulates erythroid maturation through a TP53-dependent mechanism in human CD34+ hematopoietic progenitor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 248.

Collaborative effect of Csnk1a1 haploinsufficiency and mutant p53 in Myc induction can promote leukemic transformation

AbstractIt is still not fully understood how genetic haploinsufficiency in del(5q) myelodysplastic syndrome (MDS) contributes to malignant transformation of hematopoietic stem cells. We asked how compound haploinsufficiency for Csnk1a1 and Egr1 in the common deleted region on chromosome 5 affects hematopoietic stem cells. Additionally, Trp53 was disrupted as the most frequently comutated gene in del(5q) MDS using CRISPR/Cas9 editing in hematopoietic progenitors of wild-type (WT), Csnk1a1–/+, Egr1–/+, Csnk1a1/Egr1–/+ mice. A transplantable acute leukemia only developed in the Csnk1a1–/+Trp53–edited recipient. Isolated blasts were indefinitely cultured ex vivo and gave rise to leukemia after transplantation, providing a tool to study disease mechanisms or perform drug screenings. In a small-scale drug screening, the collaborative effect of Csnk1a1 haploinsufficiency and Trp53 sensitized blasts to the CSNK1 inhibitor A51 relative to WT or Csnk1a1 haploinsufficient cells. In vivo, A51 treatment significantly reduced blast counts in Csnk1a1 haploinsufficient/Trp53 acute leukemias and restored hematopoiesis in the bone marrow. Transcriptomics on blasts and their normal counterparts showed that the derived leukemia was driven by MAPK and Myc upregulation downstream of Csnk1a1 haploinsufficiency cooperating with a downregulated p53 axis. A collaborative effect of Csnk1a1 haploinsufficiency and p53 loss on MAPK and Myc upregulation was confirmed on the protein level. Downregulation of Myc protein expression correlated with efficient elimination of blasts in A51 treatment. The “Myc signature” closely resembled the transcriptional profile of patients with del(5q) MDS with TP53 mutation.

Forward Genetic Screen Implicates Drivers of Leukemic Progression in a Novel Model of Trp53 R270H myelodysplastic Syndrome

Myelodysplastic syndrome (MDS) is characterized by bone marrow failure and a highly variable clinical course. The most catastrophic complication of MDS is transformation to secondary acute myeloid leukemia (sAML). Notably, mutations in TP53 confer the single highest risk of transformation to sAML and death. However, some patients with TP53 mutated MDS do not develop sAML, suggesting that additional genetic events cooperate with TP53 mutations to transform MDS to sAML. Understanding the mechanisms of transformation of MDS to sAML could provide targets for therapeutic intervention. To model the genetics of MDS, we crossed mice bearing Trp53 R270H ( Trp53 is the murine TP53 gene) and deletion of genes syntenic with human chromosome 5q (del(5q)). To discover how additional mutations contribute to disease progression, we utilized Sleeping Beauty (SB) transposon mutagenesis in Trp53 R270H/del(5q) mice. SB transposase mobilized SB mutagenic T2/Onc transposons which randomly insert within the genome. T2/Onc transposons are designed to induce gain or loss of function alterations depending on the site and orientation of insertion with respect to targeted genes. We used the Mx1-Cre transgene to activate SB transposase and T2/Onc transposition in hematopoietic progenitors. Trp53 R270H anddel(5q)(or cytogenetically normal, CN) mice were crossed to SB mice to generate donor mice of the following genotypes: Trp53 R270H/del(5q)/ SB, Trp53 R270H/CN /SB, Trp53 WT/del(5q)/ SB, Trp53 WT/CN/ SB mice, and mice without SB transposition, (no transposition, NT: Trp53 R270H/del(5q) /NT). Bone marrow cells were transplanted into recipients, and SB insertional mutagenesis was activated using pI-pC to activate Cre. Mice receiving Trp53 WT/CN/ SB bone marrow developed more frequent T-cell leukemia (n=3/10) than myeloid leukemia (n=1/10). In contrast, mice receiving Trp53 R270H/del(5q)/SB and Trp53 R270H/CN /SB bone marrow developed predominantly myeloid leukemia (n=14/28) more commonly than T-cell leukemia (1/28). Mixed phenotype leukemia was seen in 7/28 of these mice. Together, these data demonstrate a strong bias towards myeloid disease in SB-mutagenized Trp53 R270H bone marrow. To identify genes with SB insertions, we performed RNA sequencing to detect SB T2/Onc transposon-endogenous genefusion transcripts. Among Trp53 WT/CN/ SB leukemias, the most common recurrent SB fusions involved Notch1 and Ikzf1 as has previously reported for SB-associated T-cell leukemias . Among Trp53 R270H/del(5q)/SB and Trp53 R270H/CN /SB leukemias, the most common recurrent SB-fusions involved Erg, Eras and Il2rb with Erg fusions detected 85% of Trp53 R270H leukemias (n=17/20). SB inserted upstream of Erg promoter indicating that these fusions likely upregulate expression of Erg. Indeed, Erg levels are significantly higher in leukemias that express SB-Erg fusions relative to leukemias that do not (p<0.0023). ERG is not recurrently mutated in human AML, but the ERG gene locus is commonly amplified, especially TP53 mutant AML. ERG is known to support normal hematopoietic stem cell self-renewal. Notably, Erg-insertions were also detected in a model of MDS expressing stabilized cyclin E with SB-mediated progression to erythroleukemia (Loeb 2019). Using gene set enrichment analysis, we found that hematopoietic stem cell and leukemic stem cell signatures are enriched in Erg-SB fusion leukemias. In our analyses of two independent data sets (TCGA and BEAT AML), stem cell signatures are also among the most highly enriched pathways in human AMLs expressing high ERG levels. Furthermore, in a human AML single cell RNA sequencing dataset (van Galen 2019), we found that ERG expression is highest in AML cells with the most immature stem and progenitor-like features. Together, these findings implicate a role for ERG as a driver of progression of MDS to AML by enhancing aberrant self-renewal. In summary, we present a novel murine model of Trp53/del(5q) MDS. In this model, Erg upregulation is associated with progression to AML and upregulation of leukemia stem cell gene expression profiles. These data implicate ERG as a major contributor to progression of MDS to secondary AML in the setting of mutant p53. Understanding the mechanisms of disease progression and self-renewal in myeloid malignancies with p53 mutations is critical to define effective therapeutic strategies in these rapidly fatal, treatment resistant diseases.

Lenalidomide derivatives and proteolysis-targeting chimeras for controlling neosubstrate degradation

Lenalidomide, an immunomodulatory drug (IMiD), is commonly used as a first-line therapy in many haematological cancers, such as multiple myeloma (MM) and 5q myelodysplastic syndromes (5q MDS), and it functions as a molecular glue for the protein degradation of neosubstrates by CRL4CRBN. Proteolysis-targeting chimeras (PROTACs) using IMiDs with a target protein binder also induce the degradation of target proteins. The targeted protein degradation (TPD) of neosubstrates is crucial for IMiD therapy. However, current IMiDs and IMiD-based PROTACs also break down neosubstrates involved in embryonic development and disease progression. Here, we show that 6-position modifications of lenalidomide are essential for controlling neosubstrate selectivity; 6-fluoro lenalidomide induced the selective degradation of IKZF1, IKZF3, and CK1α, which are involved in anti-haematological cancer activity, and showed stronger anti-proliferative effects on MM and 5q MDS cell lines than lenalidomide. PROTACs using these lenalidomide derivatives for BET proteins induce the selective degradation of BET proteins with the same neosubstrate selectivity. PROTACs also exert anti-proliferative effects in all examined cell lines. Thus, 6-position-modified lenalidomide is a key molecule for selective TPD using thalidomide derivatives and PROTACs.

Foxm1 haploinsufficiency drives clonal hematopoiesis and promotes a stress-related transition to hematologic malignancy in mice.

Clonal hematopoiesis plays a critical role in the initiation and development of hematologic malignancies. In patients with del(5q) myelodysplastic syndrome (MDS), the transcription factor FOXM1 is frequently downregulated in CD34+ cells. In this study, we demonstrated that Foxm1 haploinsufficiency disturbed normal hematopoiesis and conferred a competitive repopulation advantage for a short period. However, it impaired the long-term self-renewal capacity of hematopoietic stem cells, recapitulating the phenotypes of abnormal hematopoietic stem cells observed in patients with MDS. Moreover, heterozygous inactivation of Foxm1 led to an increase in DNA damage in hematopoietic stem/progenitor cells (HSPCs). Foxm1 haploinsufficiency induced hematopoietic dysplasia in a mouse model with LPS-induced chronic inflammation and accelerated AML-ETO9a-mediated leukemogenesis. We have also identified Parp1, an important enzyme that responds to various types of DNA damage, as a target of Foxm1. Foxm1 haploinsufficiency decreased the ability of HSPCs to efficiently repair DNA damage by downregulating Parp1 expression. Our findings suggest that the downregulation of the Foxm1-Parp1 molecular axis may promote clonal hematopoiesis and reduce genome stability, contributing to del(5q) MDS pathogenesis.

Inactivation of p53 provides a competitive advantage to del(5q) myelodysplastic syndrome hematopoietic stem cells during inflammation.

Inflammation is associated with the pathogenesis of myelodysplastic syndromes (MDS) and emerging evidence suggests that MDS hematopoietic stem and progenitor cells (HSPC) exhibit an altered response to inflammation. Deletion of chromosome 5 (del(5q)) is the most common chromosomal abnormality in MDS. Although this MDS subtype contains several haploinsufficient genes that impact innate immune signaling, the effects of inflammation on del(5q) MDS HSPC remains undefined. Utilizing a model of del(5q)-like MDS, inhibiting the IRAK1/4-TRAF6 axis improved cytopenias, suggesting that activation of innate immune pathways contributes to certain clinical features underlying the pathogenesis of low-risk MDS. However, low-grade inflammation in the del(5q)-like MDS model did not contribute to more severe disease but instead impaired the del(5q)-like HSPC as indicated by their diminished numbers, premature attrition and increased p53 expression. Del(5q)-like HSPC exposed to inflammation became less quiescent, but without affecting cell viability. Unexpectedly, the reduced cellular quiescence of del(5q) HSPC exposed to inflammation was restored by p53 deletion. These findings uncovered that inflammation confers a competitive advantage of functionally defective del(5q) HSPC upon loss of p53. Since TP53 mutations are enriched in del(5q) AML following an MDS diagnosis, increased p53 activation in del(5q) MDS HSPC due to inflammation may create a selective pressure for genetic inactivation of p53 or expansion of a pre-existing TP53-mutant clone.

Treatment Patterns and Outcomes in Lower-Risk, Non-Del(5q) Myelodysplastic Syndromes: Findings from a Global Real-World Medical Record Review Study

Background: Myelodysplastic syndromes (MDS) are a group of clonal hematopoietic stem cell malignancies defined by ineffective hematopoiesis and peripheral blood cytopenias. Patients often present with anemia and frequently require red blood cell transfusions (RBCTs), which may lead them to become transfusion-dependent (TD). This results in poor quality of life and reduced survival. In patients with non-del(5q) lower-risk (LR)-MDS, the standard of care includes erythropoiesis-stimulating agents (ESAs) ± granulocyte colony-stimulating factor as first-line (1L) therapy; however, although clinical trials have demonstrated efficacy, there is little evidence from real-world settings. This study aimed to assess real-world data to investigate treatment patterns and prevailing hematologic outcomes in patients with non-del(5q) LR-MDS. Methods: In this retrospective medical record review, eligible patients ≥ 18 years of age diagnosed with non-del(5q) LR-MDS between July 1, 2013 and September 30, 2018 were identified by participating hematologist-oncologists in the US, Canada (CAN), UK, and EU (France, Germany, Spain). Demographics, clinical characteristics, treatment patterns, and hematologic outcomes data were abstracted (June-November 2021) and reported descriptively. Aggregated analyses were performed using pooled US/CAN and UK/EU data sets for the overall cohort and for patient subgroups treated with clinician-defined 1L and second-line (2L) ESA-containing regimens. Results: Medical record data were abstracted for 119 patients with non-del(5q) LR-MDS in US/CAN (median age, 61.5 years; 71.4% male) and 245 patients in UK/EU (median age, 67.3 years; 71.4% male). Median follow-up duration from MDS diagnosis was 50.5 months (US/CAN) and 67.8 months (UK/EU). At initial diagnosis, 50.4% (US/CAN) and 53.5% (UK/EU) of patients had Low-risk MDS (per Revised International Prognostic Scoring System). Of the 71 patients in US/CAN and 123 patients in UK/EU who were tested successfully for the SF3B1 mutation, 16.9% (US/CAN) and 15.5% (UK/EU) were positive. Most of the patients initiated 1L therapy (US/CAN: 109 [91.6%]; UK/EU: 225 [91.8%]) and more than one-third initiated a 2L therapy (US/CAN: 45 [37.8%]; UK/EU: 89 [36.3%]). In 1L, 2.8% patients in US/CAN and 2.2% in UK/EU received treatment with lenalidomide. Most patients were treated with ESA-containing regimens in 1L, with a median duration of therapy of ≥ 2 years (US/CAN: 97 [89.0%], of which 86.6% was ESA monotherapy; UK/EU: 203 [90.2%], of which 85.7% was ESA monotherapy). Treatment characteristics and hematologic outcomes for patients treated with ESAs are presented in the Table. Among patients who were TD before 1L ESA treatment (36 [37.1%] US/CAN; 104 [51.2%] UK/EU), a small proportion (2.8% US/CAN; 14.4% UK/EU) became transfusion independent (TI) during 1L; RBCT reduction threshold (≥ 50%) was met by 41.7% in US/CAN (median duration, 23 months) and 52.9% in UK/EU (median duration, 13 months). Among patients treated with ESA-containing regimens in 2L (12 [26.7%] US/CAN; 26 [29.2%] UK/EU), most had been treated with ESA in 1L (US/CAN: 10 [83.3%]; UK/EU: 22 [84.6%]). The most frequently reported reasons for reinitiating treatment with ESAs in 2L were overall health status (54.6% US/CAN; 40.9% UK/EU), treatment efficacy (45.5% US/CAN; 63.6% UK/EU), and disease characteristics (36.4% US/CAN; 45.5% UK/EU). In the subgroup of patients who reinitiated ESA in 2L, the most frequently reported reasons for discontinuation of 1L ESA were patient decision (45.5% US/CAN; 22.7% UK/EU), completion of planned course of therapy (45.5% US/CAN; 18.2% UK/EU), and progressive disease (36.4% US/CAN; 68.2% UK/EU). Conclusions: In this real-world study, the most frequently used treatments for the management of anemia in patients with non-del(5q) LR-MDS were ESA-containing regimens. A considerable proportion of patients were TD before initiating 1L ESA therapy. For those who were re-treated with ESAs in 2L, the most common reasons for treatment reinitiation were health status, disease characteristics, and reported treatment efficacy, although only a small fraction of patients who were TD at 1L ESA initiation became TI. These findings highlight an unmet need in anemia management among patients with non-del(5q) LR-MDS, and a requirement for new and more effective treatment options. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

MDS-454 and Mutations Might Play a Role in Poor Prognosis and AML Progression in Patients With Isolated del(5q) MDS

Myelodysplastic syndromes (MDS) with isolated del(5q) represent a subtype defined by WHO with good prognosis. However, several studies have shown that the presence of somatic mutation, such as TP53, might have an impact on prognosis. In addition, recent publications suggest a role for SF3B1 in the poor outcome of some del(5q) MDS patients. Our aim was to validate the poor prognosis of SF3B1 mutations, present in patients with del(5q) MDS and to identify mutational profiles that could promote AML progression of patients del(5q) MDS. We performed a retrospective study including MDS patients with both isolated del(5q) and del(5q) with another alteration different from chromosome 7. We included 50 patients and a control group of 123 low-risk MDS patients with normal karyotype. Next-generation sequencing (NGS) data from bone marrow samples were analyzed using an in-house custom panel (>100 myeloid genes). Median age at diagnosis was similar in del(5q) MDS and control group (72.9 and 71.8 years, respectively, p>0.05). The number of mutations per patient was significantly higher in the control group than in del(5q) (a mean of 3.16 against 1.7 for del(5q), p<0.0001). Despite a higher prevalence of somatic mutations in the control group, no differences were found in median overall survival (OS) (64.5 and 60.1 months for del(5q) MDS and control group respectively, p=0.413). However, when analyzing del(5q) patients individually, OS was significantly lower in those patients with either TP53 or SF3B1 mutation (64.5 vs. 28 months median) (p=0.031). Regarding progression to AML, patients with TP53 and/or DNMT3A mutations (n=14) had a shorter time to progression compared to patients without mutations in these genes, with a median of 13.6 months vs. 26.97 months (p=0.004). Median follow-up for these patients was 14.93 (0-170.30) months. MDS patients with del(5q) have a good prognosis and a higher time to progression to AML compared to other MDS subgroups. However, the mutational profile may modify this evolution. In our study, mutations in SF3B1 and DNMT3A may be involved in OS and transformation to AML (respectively), but a larger cohort of patients is needed to confirm that.

MDS-450 Impact of Number of Cells Harboring Deletion 5q Abnormality by Conventional Cytogenetics in Isolated Deletion 5q Myelodysplastic Syndromes

Isolated del 5q myelodysplastic syndrome (MDS) is defined as presence of del 5q +/- one additional chromosomal abnormality without excess myeloblasts. Lenalidomide (len) is current standard of care for those patients. The impact of percentage of cells (number of metaphases) harboring del 5q by karyotype is not known. We examined our cohort of isolated del 5q MDS patients at Moffitt Cancer Center. We assessed baseline characteristics, response to len, and outcomes based on % of cells/metaphases harboring del 5q using G-banding "conventional cytogenetics clone size." We divided patients into 2 groups based on number of cells harboring del 5q, those with <50% i.e less than 10 out 20 metaphases and 50-100% (10 or more out of 20 metaphases). We identified 142 isolated del 5q MDS patients. Baseline characteristics of the whole cohort was similar to what has been previously described in the literature. Patients with >50% metaphases harboring del 5q had more severe anemia (Hgb < 8.0 g/dl) (30% versus 13%) (p=0.05). Among patients who were treated with len (n=100), 50% achieved a response. There was a trend for higher response rate among patients with >50% del 5q metaphases with (42/80) 53% response rate compared to (8/20) 40% among those with <50%. (p=0.32). There was no difference in presence of TP53 somatic mutation based on % of metaphases harboring del 5q. There was a non-statistically significant trend for better overall survival among patients with >50% cells with detected del 5q compared to those with <50%, median OS 57 mo versus 35 mo, p=0.90. The number of cells harboring del 5q among isolated del 5q MDS patients using regular karyotyping "clone size by conventional cytogenetics" correlated with severity of anemia, trend for better response to len and trend for better overall survival. Those findings should be further confirmed in larger cohort of patients.

Studies of a mosaic patient with DBA and chimeric mice reveal erythroid cell–extrinsic contributions to erythropoiesis

AbstractWe follow a patient with Diamond-Blackfan anemia (DBA) mosaic for a pathogenic RPS19 haploinsufficiency mutation with persistent transfusion-dependent anemia. Her anemia remitted on eltrombopag (EPAG), but surprisingly, mosaicism was unchanged, suggesting that both mutant and normal cells responded. When EPAG was withheld, her anemia returned. In addition to expanding hematopoietic stem/progenitor cells, EPAG aggressively chelates iron. Because DBA anemia, at least in part, results from excessive intracellular heme leading to ferroptotic cell death, we hypothesized that the excess heme accumulating in ribosomal protein-deficient erythroid precursors inhibited the growth of adjacent genetically normal precursors, and that the efficacy of EPAG reflected its ability to chelate iron, limit heme synthesis, and thus limit toxicity in both mutant and normal cells. To test this, we studied Rpl11 haploinsufficient (DBA) mice and mice chimeric for the cytoplasmic heme export protein, FLVCR. Flvcr1-deleted mice have severe anemia, resembling DBA. Mice transplanted with ratios of DBA to wild-type marrow cells of 50:50 are anemic, like our DBA patient. In contrast, mice transplanted with Flvcr1-deleted (unable to export heme) and wild-type marrow cells at ratios of 50:50 or 80:20 have normal numbers of red cells. Additional studies suggest that heme exported from DBA erythroid cells might impede the nurse cell function of central macrophages of erythroblastic islands to impair the maturation of genetically normal coadherent erythroid cells. These findings have implications for the gene therapy of DBA and may provide insights into why del(5q) myelodysplastic syndrome patients are anemic despite being mosaic for chromosome 5q deletion and loss of RPS14.

Genetic barcoding systematically compares genes in del(5q) MDS and reveals a central role for CSNK1A1 in clonal expansion

AbstractHow genetic haploinsufficiency contributes to the clonal dominance of hematopoietic stem cells (HSCs) in del(5q) myelodysplastic syndrome (MDS) remains unresolved. Using a genetic barcoding strategy, we performed a systematic comparison on genes implicated in the pathogenesis of del(5q) MDS in direct competition with each other and wild-type (WT) cells with single-clone resolution. Csnk1a1 haploinsufficient HSCs expanded (oligo)clonally and outcompeted all other tested genes and combinations. Csnk1a1−/+ multipotent progenitors showed a proproliferative gene signature and HSCs showed a downregulation of inflammatory signaling/immune response. In validation experiments, Csnk1a1−/+ HSCs outperformed their WT counterparts under a chronic inflammation stimulus, also known to be caused by neighboring genes on chromosome 5. We therefore propose a crucial role for Csnk1a1 haploinsufficiency in the selective advantage of 5q-HSCs, implemented by creation of a unique competitive advantage through increased HSC self-renewal and proliferation capacity, as well as increased fitness under inflammatory stress.

Therapeutic Outcomes and Prognostic Impact of Gene Mutations Including TP53 and SF3B1 in Patients with Del(5q) Myelodysplastic Syndromes (MDS)

BackgroundGenetic alterations are increasingly being recognized to play an important role in both diagnosis and prognosis of MDS. In general, MDS patients with SF3B1 mutations (MT) are known to have favorable outcomes whereas those with TP53 mutations have dismal survivals. However, it is unclear if the impact of these mutations applies to all subtypes of MDS including del(5q) which is known for its response to lenalidomide and better prognosis. Materials and MethodsWe retrospectively reviewed 132 del(5q) MDS patients who were treated at the Moffitt Cancer Center (2001-2019). ResultsAmong patients who received lenalidomide (n = 98), 50%, 42.9%, and 7.1% achieved hematologic improvement or better, no response, and disease progression/death with a median overall survival (mOS) of 93.2, 72.4, and 25.6 months, respectively (P < .0001). The mOS was 73.3 months but only 25.6 months after patients stopped lenalidomide. TP53 was the most common mutation accounting for 23.8% of the patients. Of the 63 patients with molecular data available, 23.8% harbored TP53 MT and 10% with SF3B1 MT. TP53 status did not impact OS (MT 86.4 vs. wild-type (WT) 73.3 months; P = .72) but those with SF3B1 mutations had a significantly shorter mOS compared to WT (23.9 vs. 83.5 months; P = .001). Multivariate analysis confirmed lenalidomide response and SF3B1 mutations are independently associated with outcomes. ConclusionOur findings indicate many del(5q) MDS patients will benefit from lenalidomide but survival after its failure is limited. Mutations known to have prognostic impact in MDS at large may not have the same implications in the del(5q) subset.

Research Progress of Thalidomide and Its Derivatives in Treatment of Myelodysplastic Syndrome--Review

Thalidomide and its derivatives have been used in the treatment of myelodysplastic syndrome (MDS) because of their anti-angiogenic and immunomodulatory effects. In recent years, some studies have found that thalidomide and its derivatives not only showed significant efficacy in lower-risk MDS patients with del (5q), but also showed advantages in non-del (5q) MDS patients. In addition, the discovery of its molecular targets and new substrates makes it possible to develop a new generation of immunomodulatory drugs (IMiDs) and to design IMiDs-based proteolysis-targeting chimeras. In this review, the new progress in mechanism and clinical application of thalidomide and its derivatives were summarized briefly, so as to provide a more scientific, reasonable and effective scheme to the treatment of MDS.

Dual pyroptotic biomarkers predict erythroid response in lower-risk non-del(5q) myelodysplastic syndromes treated with lenalidomide and recombinant erythropoietin.

Not available.

Lenalidomide for anemia correction in lower-risk del(5q) myelodysplastic syndrome patients of Asian ethnicity.

BackgroundTo estimate real-world outcomes in East Asian populations, we conducted a nationwide retrospective analysis of the efficacy and safety of lenalidomide for del(5q) myelodysplastic syndrome (MDS) patients with transfusion-dependent anemia in Korea.MethodsPatients aged ≥19 years who had received lenalidomide for the treatment of lower-risk, red blood cell (RBC) transfusion-dependent del(5q) MDS were selected. A filled case report form (CRF) with information from electronic medical records was requested from members of the acute myeloid leukemia (AML)/MDS Working Party of the Korean Society of Hematology. All the CRFs were gathered and analyzed.ResultsA total of 31 patients were included in this study. Of 28 evaluable patients, 19 (67.9%) achieved RBC transfusion independence (RBC-TI). Female sex and the development of thrombocytopenia during treatment were associated with achieving RBC-TI. The most common non-hematologic toxicities were pruritus, fatigue, and rashes. All non-hematologic toxicities of grades ≥3 were limited to rash (12.9%) and pruritus (6.5%). Dose reduction was required in 15 of the 19 responders (78.9%). The most common final stable dosing schedule for the responders was 5 mg once every other day (31.6%).ConclusionLenalidomide efficacy and tolerability were similar in the Asian del(5q) MDS patients and western patients. Dose reduction during treatment was common, but it was not associated with inferior outcomes.

British Society for Haematology guidelines for the management of adult myelodysplastic syndromes.

British Society for Haematology guidelines for the management of adult myelodysplastic syndromes.

Tifab Deficiency Protects Against Stress-Induced Bone Marrow Failure at the Expense of Pre-Leukemic Selection

Interstitial deletion of a single copy of chromosome 5q is the most frequent cytogenetic alteration in Myelodysplastic Syndromes (MDS), which results in reduced dosage of numerous genes. TRAF-interacting protein with forkhead-associated domain B (TIFAB) resides within the proximal commonly-deleted region on band 5q31.1, and belongs to a family of forkhead-associated domain proteins. TIFAB is deleted in nearly all reported cases of del(5q) MDS and AML. As expected, TIFAB expression is significantly lower in CD34+ and BM mononuclear cells isolated from MDS patients with del(5q) as compared with cells from MDS patients diploid at chr 5q. Recently we have shown that hematopoietic-specific deletion of Tifab in mice results in progressive BM and blood defects, including aberrant HSPC proportions, altered myeloid differentiation, and progressive cytopenia (Varney and Niederkorn et al., JEM 2015). Gene expression analysis of Tifab KO lineage-Sca1+cKit+ (LSK) cells identified dysregulation of immune-related signatures, hypersensitivity to Toll-like receptor stimulation, and enrichment of a p53 regulatory gene-set.Importantly, p53 is implicated in the pathogenesis of del(5q) MDS: 1) BM cells from murine models and BM from del(5q) patients exhibit increased p53 activity, which is thought to contribute to ineffective hematopoiesis and anemia; and 2) del(5q) MDS patients often acquire concurrent TP53 mutations that result in rapid transformation to AML and poor treatment response. To examine the effects of TIFAB on p53 function, we examined p53 target genes in TIFAB-overexpressing and -deficient cells. Gene expression profiling and qRT-PCR analysis of Tifab KOHSPCs revealed significant upregulation of p53 regulatory genes. In contrast, overexpression of TIFAB in a p53-competent cell line reduced the expression of p53 target gene, p21 . We also observed increased p53 protein in Tifab KO total BM, lineage- BM, and spleen. At steady-state and after in vitro treatment with 2 Gy irradiation, Tifab KO lineage- BM cells exhibit increased p53 protein compared to WT cells.To test whether this increased p53 stability renders Tifab -deficient HSPCs more sensitive to stress, we treated Tifab KO HSPCs both in vivo and ex vivo using irradiation. When treated with low dose irradiation ex vivo, Tifab KO HSPCs initially exhibit significantly reduced colony forming ability; however, upon secondary re-plating, Tifab KO HSPCs strikingly form more colonies than WT HSPC, resembling a clonal selection event. To model this effect in vivo, we transplanted WT or Tifab KO BM into lethally-irradiated recipient mice. After 4 weeks of hematopoietic reconstitution, recipient mice were treated with 6.5 Gy sublethal irradiation. We find that Tifab KO recipient mice are less likely to succumb to IR-induced bone marrow failure as compared to mice transplanted with WT BM cells. Instead, after 3+ months, the remaining Tifab KO recipient mice have an increased tendency to develop hematologic malignancies, evident by myeloid blasts. Collectively, these data suggest that TIFAB-deficiency protects against cellular stress by eliminating “less fit” HSPCs, at the expense of selecting for pre-leukemic populations, paralleling observations in patients with transformed MDS.To investigate the mechanism of this phenomenon, we performed a tandem-affinity tag purification and mass-spectrometry analysis of TIFAB complexes in a del(5q) AML cell line (HL60), and identified unique TIFAB-interacting proteins. The top interacting candidate was an ubiquitin-specific peptidase (USP), USP15. USPs play a major role in ubiquitin-dependent processes including DNA damage response signaling, protein degradation, and kinase activation. Specifically, USP15 has been shown to promote p53 degradation via deubiquitination and stabilization of its major negative regulator, MDM2. Through biochemical assays and a series of deletion mutants, we confirmed that TIFAB interacts with USP15 and enhances the deubiquitination of biological USP15 substrates, including MDM2. Thus, our data implicate TIFAB as an important negative regulator of p53 and modulator of USP15 DUB activity.Altogether, our findings identify a novel role for TIFAB as a modifier of p53 function, potentially via a USP15-MDM2 axis. These findings have important implications in the potential role of TIFAB and p53 signaling in the pathogenesis of del(5q) MDS and transformation to AML. DisclosuresNo relevant conflicts of interest to declare.