Del 5q MDS Research Articles

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 A012: Targeting proteasome vulnerabilities for the treatment of monosomy 7 associated blood disorders

Abstract Monosomy 7 is among the most frequent cytogenetic abnormalities in hematological disorders and is associated with short survival and drug resistance. Despite its high prevalence and detrimental impact, the therapeutic vulnerabilities underlying monosomy 7-associated blood disorders remain largely elusive, impeding progress toward improved patient care. The homeostatic cellular requirement for a normal dosage of essential genes creates an opportunity to target vulnerabilities that arise due to reduced levels of proteins encoded by a haploinsufficient essential gene. Briefly, the loss of one copy of a dosage-sensitive essential gene (gene X), in combination with the inhibition of itself or a related gene (gene Y), or an associated pathway results in lethal consequences for cells. The remarkable and selective clinical efficacy of lenalidomide for the treatment of del(5q) MDS has demonstrated how allelic haploinsufficiency underlies the sensitivity to this drug by synthetic lethality. Differential expression analysis of gene and protein expression in primary AML samples with monosomy 7 revealed significant downregulation of multiple proteasome pathway members at the protein level, but not at the RNA level. Primary AML samples with -7/del(7q) exhibited increased sensitivity (low IC50) to the proteasome inhibitor bortezomib, as evidenced by two independent ex vivo drug screening cohorts (the Beat AML and the FIMM study). Chromosome 7 harbors four proteasome subunits, PSMA2, PSMC2, PSMG3, and SEM1. We performed gene expression, protein expression, copy number analysis, and individual gene knockout experiments. The results have revealed PSMA2 to be a haploinsufficient essential gene on chromosome 7. PSMA2 knockout confers leukemia a growth disadvantage for multiple AML cell clines in both TP53 wild-type and knockout backgrounds. We generated PSMA2 isogenic hemizygous deletion and diploid single-cell clones. PSMA2 hemizygous deletion cells exhibited approximately half the protein expression compared to diploid controls, confirming that PSMA2 is a haploinsufficient gene. PSMA2hemizygous deletion single-cell clones showed significantly enhanced sensitivity to all three evaluated proteasome inhibitors (bortezomib, ixazomib, and carfilzomib), aligning with the sensitivity observed in primary -7/del(7q) leukemia samples. PSMA2 hemizygous deletion cell clones displayed increased p38 and decreased pERK levels upon treatment with proteasome inhibitors, potentially contributing to their increased sensitivity to proteasome inhibitors. Proteomics analysis and in vivo validation is ongoing. As such, we have identified haploinsufficient essential gene PSMA2 mediated proteasome pathway vulnerability in monosomy 7 associated leukemia and further showed that proteasome inhibitors as promising therapeutic approaches for treating hematological disorders associated with monosomy 7. Citation Format: Haijiao Zhang, Basil Allen, Daniel Bottomly, Peter Ryabinin, Schannon K. Targeting proteasome vulnerabilities for the treatment of monosomy 7 associated blood disorders [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr A012.

The Transcriptional Impact of 5q Deletion in MDS at Single Cell Resolution

Introduction: Myelodysplastic syndromes with 5q deletion (del(5q) MDS) are characterized by two commonly deleted regions (CDRs): 5q31 and 5q32-33. Although several genes within these CDRs have been associated with disease pathophysiology, the molecular basis underlying its origin is still unknown. Lenalidomide represents the first therapeutic approach for del(5q) MDS, but patients experience loss of responsiveness within 2-3 years, highlighting the need of characterizing the mechanisms impairing treatment response. While previous studies have identified dysregulated genes and pathways in the disease, they overlooked that the hematopoietic system of these patients is composed of a mixture of cells with and without the deletion (del(5q) and non-del(5q) cells), which hampers the ability to unravel the real transcriptional impact of the deletion. Methods: We isolated by FACS and performed single-cell RNA sequencing (scRNAseq, 10XGenomics Chromium platform) of CD34+ cells from 4 untreated patients, 2 patients treated with lenalidomide (partial (PCR) and complete (CCR) cytogenetic responders), and 3 healthy elderly donors. To identify del(5q) and non-del(5q) cells, the copy number alteration (CNA) inference methods CopyKat and CaSpER were applied. To unveil the transcriptional alterations prompted by the deletion, differential expression (DE), gene regulatory network (GRN) and cell-to-cell communication (CCC) analyses were performed (Figure 1). Results: CNA methods robustly identified del(5q) cells and non-del(5q) cells, which was consistent with the del(5q) percentage obtained by karyotyping. At diagnosis, del(5q) cells were detected in all hematopoietic progenitors, including the hematopoietic stem cells (HSC), suggesting that the deletion occurs in the earliest stages of hematopoietic differentiation. Although the distribution of del(5q) cells was highly heterogeneous among patients (Figure 2), a positive enrichment of del(5q) cells was detected in erythroid progenitors, underscoring the role of this genetic lesion in the anemia characterizing del(5q) MDS. To delve into the transcriptional mechanisms differentiating del(5q) and non-del(5q) cells, we performed DE and GRN analyses. Only 7 genes were downregulated in del(5q) cells; however, most play a key role in MDS and other tumors, such as PRSS21, MAP3K7CL, and CCL5. GRN analyses identified regulons JARID2, IRF1 and KAT6B, known to be key for proper hematopoietic differentiation, showing less activity in del(5q) cells. Likewise, the regulons RERE and KDM2A, which downregulated genes involved in ribosomal processes, were more active in del(5q) cells, supporting the concept that cells harboring the deletion may have a more prominent role in promoting altered hematopoiesis. However, comparisons against healthy donor cells suggested that non-del(5q) cells could share similar, albeit attenuated, abnormal behavior to del(5q) cells. In line with these results, CCC analyses identified minimal differential interactions in del(5q) cells with respect to non-del(5q) cells, indicating that the deletion does not confer a differential communicative potential to the cells. Importantly, several interactions present in MDS cells were absent in healthy cells, such as the AGTRAP-RACK1, whose members have been postulated as potential therapeutic targets for promoting proliferation in other cancers. After lenalidomide, del(5q) and non-del(5q) cells from PCR and CCR patients showed higher protein degradation and erythropoietin signaling, as well as higher activity of the PD-L1/PD-1 pathway, suggesting a potential immunosuppressive mechanism of these cells after treatment. Even if several transcriptional alterations detected at diagnosis were reverted after lenalidomide, ribosomal translation processes were still altered, even at time of CCR. These results evidence that, after lenalidomide, non-del(5q) progenitor cells revert some of the transcriptional alterations that present at diagnosis, while maintaining other lesions that could be relevant for abnormal hematopoiesis, and potentially, for the future relapse of the patients. Conclusions: This study provides the first characterization of del(5q) and non-del(5q) cells at the single-cell resolution and reveals previously unknown transcriptional alterations that could contribute to disease pathogenesis or less responsiveness to lenalidomide.

Lenalidomide treatment of Japanese patients with myelodysplastic syndromes with 5q deletion: a post-marketing surveillance study

Lenalidomide was approved in Japan for the treatment of patients with myelodysplastic syndromes associated with a 5q deletion (del 5q-MDS) in August 2010. A post-marketing surveillance (PMS) study enrolled 173 patients with del 5q-MDS who started lenalidomide treatment between August 2010 and September 2011 (mean ± standard deviation [SD] age 72.4 ± 9.0 years) and observed for up to 6 cycles or 6 months. Adverse drug reactions (ADRs) and serious ADRs were reported in 78.0% and 50.9% of patients. The most commonly observed ADRs were thrombocytopenia or platelet count decreased (46.2%), neutropenia or neutrophil count decreased (42.2%), and rash (23.1%). Of 114 patients who were red blood cell transfusion-dependent at baseline, 39 (34.2%) achieved transfusion independence during lenalidomide treatment. Of 173 patients, 19 (11.0%) had confirmed acute myeloid leukemia (AML) progression during the study. Moreover, long-term follow-up (3 years) was available for 68 of the 173 patients, of whom 12 (17.6%) progressed to AML during the additional period. This PMS study investigated the safety and effectiveness of lenalidomide in patients with del 5q-MDS. No new safety concerns were noted in routine clinical use in Japan and no evidence was found for an increased risk of AML progression following lenalidomide treatment.

TP53 Allelic State in Myelodysplastic Syndromes with 5q Deletion

Introduction: Around 10% of de novo MDS harbor mutations in tumor protein p53 (TP53) gene. Recently, it has been shown that two-thirds of patients with MDS and mutated TP53 gene exhibit multi-hit mutations of TP53, this being associated with poor clinical features and outcome. Taking into consideration that MDS with isolated deletion of chromosome 5 (MDS-del5q) are particularly enriched in TP53 mutations (around 20% exhibit TP53 aberrations), we decided to analyze the TP53 allelic state in patients with MDS-del(5q). Methods: Patients from seven centers belonging to the Spanish Group of MDS (GESMD) and diagnosed with MDS-del5q according to the WHO 2017 classification were included. Therapy related MDS-del5q, as well as patients on disease modifying therapy prior the genetic analysis were excluded. Clinical characteristics and laboratory data were collected at the moment of diagnosis. Genetic profiling included conventional G-banding analyses, FISH for TP53 deletion, next-generation sequencing (NGS) analysis of a panel of 27 genes recurrently mutated in MDS (ASXL1, CALR, CBL, CEBPA, CSF3R, DNMT3A, ETV6, EZH2, FLT3, IDH1, IDH2, JAK2, KIT, KRAS, MPL, NPM1, NRAS, PTPN11, RUNX1, SETBP1, SF3B1, SRSF2, TET2, TP53, U2AF1, WT1, ZRSR2), and Sanger sequencing. Variant filtering and categorization were performed according to the Spanish Guidelines (Palomo et al., 2019). Based on TP53 mutations and allelic imbalances in chromosome 17p, patients were classified as follows (Bernard et al, 2020): 1) TP53 wild-type (TP53-wt) when no TP53 mutations were found; 2) TP53 monoallelic (TP53-monoallelic) when only 1 mutation was present; and 3) TP53 multi-hit (TP53-multihit) when multiple mutations or mutation(s) and concomitant deletion or copy number neutral loss of heterozygosity (LOH) were identified. Statistical analysis was performed by R (4.2.0). Results: We included 140 MDS-del(5q). Patient's characteristics are detailed in Table 1. Median overall survival (OS) was 96.7 months. Conventional cytogenetic analysis and/or FISH were performed in all cases. NGS was done in 76% (n=106), whereas in the remaining 24% (n=34) analysis of TP53, TET2, CBL and IDH1/2 was performed by Sanger sequencing. Eighty-six per cent (n=120) of patients were TP53-wt, and 14% (n=20) presented TP53 abnormalities: 12% (n=17) TP53-monoallelic and, interestingly, only 2% (n=3) TP53-multihit (Figure 1). In most TP53-monoallelic patients (n=10/17), TP53 mutation was detected as a single genetic event, while the most frequent concomitant mutation among the remaining MDS was DNMT3A (n=3). All TP53-multihit cases harbored two TP53 mutations, but no deletions or LOH in 17p were detected. In 2 out of these 3 patients, no other concomitant mutations were detected. As per the general mutational landscape of the MDS-del(5q) TP53-wt patients, the most frequently affected genes were SF3B1 (15%), DNMT3A (14%), TET2 (13%), ASXL1 (8%), JAK2 (4%), SETBP1 (2%) and CBL (2%). Finally, median OS was 96.7 months for MDS-del(5q) TP53-wt, 62.8 months for MDS-del(5q) TP53-monoallelic, and 56.9 months for MDS-del(5q) TP53-multihit; p= 0.3, with a trend for a worse OS of TP53-monoallelic compared with TP53-wt (HR, 0.5; p=0.106). Conclusions: This study shows that, as opposite to other MDS subgroups, the presence of multi-hit TP53 status is uncommon in patients with MDS-del5q. Larger series are currently collected to further ascertain the prognostic impact of TP53 aberrations in patients with MDS-del(5q). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Long-Term Evolution of Somatic Mutations in Patients with Del(5q) MDS Early Treated with Lenalidomide in the Sintra-Rev Clinical Trial: Safe and Effecitive Approach?

INTRODUCTION Lenalidomide (LEN) is the election treatment in del(5q) MDS, approved only in case of RBC transfusion dependency (TD). Only scarce retrospective data exploring LEN in non-TD setting are available, suggesting a benefit. SintraRev is the first randomized clinical trial evaluating LEN in del(5q) MDS with anemia but without TD. While efficacy has been confirmed in this trial, safety regarding clonal evolution and disease progression has not been addressed in this early treatment context. METHODOLOGY SintraRev is a phase III multicenter clinical trial in low-risk del(5q) MDS patients with anemia without TD. Patients were randomized (2:1) in a double-blind design to LEN (5mg/day continuously) vs placebo (PCB) for 2 years of treatment and 2 years of follow-up (FU). In addition to clinical results, we aimed to study the role of molecular profiling in this early therapeutic approach (LEN use prior to TD) in both arms during FU (baseline, week 12 and the last available FU). Next-generation sequencing (NGS) was performed in available samples. Sequential samples were collected before treatment initiation and then every 6 months until last FU. A capture-based NGS panel targeting 65 myeloid genes was used. Libraries were prepared following manufacturer's instructions (Twist BioScience) and run on Illumina sequencers. Mutations were considered at coverage of 2000x and >5% variant allele frequency (VAF). RESULTS Sixty-one patients were included in SintraRev with a median FU of 60.6 (32.3-73.9) months. A total of 47 patients (29 LEN, 18 PCB) had NGS data. At baseline, 32%, 36% and 32% of patients had 0, 1 and ≥2 mutations, respectively. More frequently mutated genes were SF3B1 (25.5% of patients) and TP53 (21%). No differences were observed regarding SF3B1 (p=0.74), TP53 (p=0.48) and DAT (DNMT3A, ASXL1 and TET2, p=1.0) mutations between groups. Furthermore, no differences in neither median number of mutations per patient (1: 0-4) nor VAF were found between LEN and PCB initially (p>0.05). At week 12, median VAF of mutations was significantly lower in the LEN group as compared to baseline (1.84% vs 9%, respectively p<0.01, n=21), but remaining stable in the PCB arm (18% w12 vs 17% baseline, p=0.594). Interestingly, in the long-term FU (median 22.4 months - 3.03 to 68.77) an increase in median VAF (from 17% to 29%, p<0.001, n=13) was observed in PCB arm (Fig 1). Clonal evolution among LEN & PCB patients during treatment was different, with a reduction in predominating clones in LEN arm, while remaining stable in the PCB group (Fig 2). In the long-term FU, both PCB & LEN showed acquisition of additional mutations (20% PCB & 22.7% LEN, p=1.0). Nevertheless, all these new mutations in the LEN group were acquired several months after loss of response to treatment or LEN discontinuation. Interestingly, dynamics of mutations under LEN treatment did not affect DAT mutations, which remained stable when cytogenetic response (CyR) was achieved. In this way, 87,5% of DAT mutations showed an independent behavior from the clone with 5q deletion. However, most SF3B1 (62,5%) and TP53 mutations (100%) were affected by LEN treatment in parallel with the disappearance of the del(5q) clone at the time of CyR. In this regard, responses were not affected by the persistence of DAT mutations (>80% of erythroid response, ER, and CyR) and interestingly, SF3B1mut patients achieved 100% of ER and CyR. In contrast, only 20% of TP53mut patients achieved ER and 60% CyR. AML evolution occurred in 13.8% and 22.2% patients in LEN & PCB arms, respectively (p=0.43). In patients transforming to AML, 50% had TP53mut in LEN while no TP53 mutations were found in PCB AML progression. No differences were found regarding median time to AML between the two groups (25.87 months, p=0.2). CONCLUSIONS MDS patients showing 5q deletion and without TD early treated with LEN did not showed more clonal evolution than the same set of patients in PCB arm. In fact, the acquisition of additional mutations in LEN patients was observed after loss of response or LEN discontinuation. Interestingly, molecular responses were confirmed in LEN patients by reduction of mutation's median VAF at week 12. Dynamic evolution of DAT alterations was not affected by LEN treatment, suggesting a role as CHIP-associated mutations. In contrast, most SF3B1 and TP53 mutations were modified in parallel with the del(5q) clone at the time of CyR. The maintenance of CHIP-related mutations did not affect either ER or CyR. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Evaluation of Lenalidomide (LEN) Vs Placebo in Non-Transfusion Dependent Low Risk Del(5q) MDS Patients. Final Results of Sintra-REV Phase III International Multicenter Clinical Trial

Background: Most MDS patients with del(5q) develop RBC TD or need treatment for symptomatic anemia early after diagnosis (median time to transfusion/treatment of 20 months, López Cadenas et al abstract 3180 ASH 2016). Lenalidomide (LEN) is a reference treatment in MDS-del(5q) but approved in many countries only when RBC-TD occurs. No randomized trials before Sintra-Rev had explored the role of LEN in the setting of non-TD anemia in del(5q) MDS. Interim analysis of Sintra-Rev (Lopez Cadenas et al abstract 536 ASH 2020) showed that early treatment with LEN prolongs the Transfusion Free survival (p=0.021), with a high percentage of erythroid (72%) and cytogenetic responses (80%) among LEN treated patients. Material: Sintra-Rev is a phase III multicenter trial in low-risk MDS-del(5q) patients with anemia without TD. Patients were randomized (2:1) in a double-blind design to LEN (5mg/day continuously) vs placebo for 2 years of treatment and 2y of follow-up. The primary endpoint was the time to TD. 2º endpoints included erythroid (HI-E), cytogenetic response (CyR), overall survival (OS), event free survival (EFS), time to AML and mutational analysis (NGS). Here, we report the final results of this clinical trial once all patients have completed their participation in the study and after extending follow-up to explore long-term AML transformation and/or death. Results: Main clinical characteristics of the 61 patients (ITT population) included are summarized in Table 1: In 32% of patients no somatic mutation was identified (NGS), compared to 36% with one mutation and 32% with ≥ 2 mutations. TP53mut was present at diagnosis in 17.2% and 27.8% of LEN and Placebo patients (p=0.48). The evaluable population included 54 patients (7 patients were excluded due either to screening failure [1 pat] or failure to complete at least 12w of treatment [6 pat] without progression disease). Median time on treatment was 66 weeks (3-121), 95w in the LEN arm and 42w in the placebo arm (p=0.392). Forty-seven percent patients in the LEN arm successfully completed the study compared to 33% in the placebo arm. After a median follow up of 60.6 months (Q1 32.3-Q3 73.9), median OS was 101.2m (95CI 54.8 - 147.7) without statistically significant differences between the two treatment groups (p=0.529). In relation to the primary objective of this trial, Time to TD (ITT-pop) was 66.3mo (95CI 37 - 95.5) for LEN patients and 11.6mo for the placebo arm (HR 0.414 95CI 0.196- 0.875, p=0.021) (Figure 1). EFS was 60.2mo (CI95% 45-74) for LEN compared to 20.2mo (CI95% 0 - 51) for Placebo (p=0.058) in the ITT Population (HR 0.46, CI95% 0.2 - 0.948, p=0.035 in the evaluable population). HI-E was observed in 70% of LEN patients (ITT-pop) compared to 0% in the placebo arm (p<0.001). Median Hemoglobin improvement in responders was 2.7 g/dL. Eighty percent of LEN patients (ITT-pop) achieved a cytogenetic response (CyR) (87.5% complete CyR) compared to 0% of patients receiving placebo (p<0.001). AML evolution was identified in 6/40 (15%) and 5/21 (23.8%) for LEN and Placebo patients respectively (p=0.488). NGS analysis during follow up showed the mean number of mutations per patient to be reduced in the LEN arm at week 12 (0,84 mutations compared to 1,21 previous to treatment; (p<0.01)), while no differences were found in the Placebo group (p=0.59). In addition, no differences were identified regarding clonal evolution for LEN and Placebo patients during study follow-up. Fifty-eight patients developed at least one adverse event (AE) during the trial (no differences between the LEN and placebo arm), related to the drug 86.8% in the LEN and 33.3% in the placebo arm, respectively (p<0.001). Severe AEs were the same as those collected in the interim analysis (abstract 536 ASH 2020). Conclusions: This final analysis of the Sintra-Rev clinical trial confirms that low dose LEN (5 mg), in anemic non-TD low risk MDS del(5q) patients, prolongs time to TD from the beginning of treatment (66.6 mo vs 11.6mo), improves Hb levels (70% of ER) and induces good quality clonal responses (80% CyR), ie potentially more responses than in historical series of MDS del(5q) treated with LEN at time of TD, with a manageable safety profile, and no increased progression rate or clonal evolution. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clinical and Biological Characterization of Low Risk MDS Patients According to Ring Sideroblasts (RS). Comparison between the 3 Subgroups in WHO2017 Classification. Study from the Spanish MDS Registry

Clinical and Biological Characterization of Low Risk MDS Patients According to Ring Sideroblasts (RS). Comparison between the 3 Subgroups in WHO2017 Classification. Study from the Spanish MDS Registry

TLR7 ligation augments hematopoiesis in Rps14 (uS11) deficiency via paradoxical suppression of inflammatory signaling

AbstractMyelodysplastic syndrome (MDS) is a hematological malignancy characterized by blood cytopenias and predisposition to acute myeloid leukemia (AML). Therapies for MDS are lacking, particularly those that have an impact in the early stages of disease. We developed a model of MDS in zebrafish with knockout of Rps14, the primary mediator of the anemia associated with del(5q) MDS. These mutant animals display dose- and age-dependent abnormalities in hematopoiesis, culminating in bone marrow failure with dysplastic features. We used Rps14 knockdown to undertake an in vivo small-molecule screening, to identify compounds that ameliorate the MDS phenotype, and we identified imiquimod, an agonist of Toll-like receptor-7 (TLR7) and TLR8. Imiquimod alleviates anemia by promoting hematopoietic stem and progenitor cell expansion and erythroid differentiation, the mechanism of which is dependent on TLR7 ligation and Myd88. TLR7 activation in this setting paradoxically promoted an anti-inflammatory gene signature, indicating cross talk via TLR7 between proinflammatory pathways endogenous to Rps14 loss and the NF-κB pathway. Finally, in highly purified human bone marrow samples from anemic patients, imiquimod led to an increase in erythroid output from myeloerythroid progenitors and common myeloid progenitors. Our findings have both specific implications for the development of targeted therapeutics for del(5q) MDS and wider significance identifying a potential role for TLR7 ligation in modifying anemia.

Lenalidomide-Mediated Erythroid Improvement in Non-Del(5q) Myelodysplastic Syndromes Is Associated with Bone Marrow Immuno-Remodeling

Introduction: The main therapeutic goal for patients with International Prognostic Scoring System (IPSS) low or intermediate risk MDS is to improve anemia. Lenalidomide yields red blood cell (RBC) transfusion independence (RBC-TI) in 60-75% of del(5q) MDS, and 40-60 % achieve complete or partial cytogenetic response.Lenalidomide also yields RBC-TI ≥8 weeks in ~25% of RBC transfusion-dependent (TD) non-del(5q) MDS patients after resistance to erythropoiesis-stimulating agent (ESA). Although reasons for lenalidomide being less effective in non-del(5q) MDS patients are unclear, it has been shown that those who responded to lenalidomide had a poor expression of erythroid genes in their bone marrow (BM).Addition of epoetin β to lenalidomide increased the erythroid response (HI-E) compared to lenalidomide alone in the GFM-LEN-EPO-2008 clinical trial (clinicaltrials.gov NCT01718379, EudraCT number 2008-008262-12), but the combination could be less efficient if epoetin β is delayed after response failure to lenalidomide alone. We have shown that the number and size of the clones within the hematopoietic stem and progenitor cell (HSPC) compartment are diminished in responding patients while the loss of response is associated with the re-increase of allele burden and/or the positive selection of clones with additional mutations. Therefore, lenalidomide efficacy may be due to a direct antitumor activity on clonal HSPCs. Interference with leukemic and stromal cells interaction and/or delivery of an immunomodulatory signal through the activation of cytotoxic T cells and NK cells have also been suggested.Methods: Appended to the GFM-LEN-EPO2008 clinical trial, we performed a transcriptomic analysis of bone marrow samples at baseline and after 4 cycles or more than 9 cycles of treatment to investigate molecular markers of response using Affymetrix U133Plus 2.0 microarrays or RNA-seq.Results: Genes differentially expressed between responders and non-responders at baseline were considered for Gene Set Enrichment Analysis (GSEA) and comprised (i) upregulated genes implicated in cell division, epigenetic modifications and translation, and (ii) downregulated genes involved in lymphocyte activation. A supervised class-prediction analysis performed by using Prediction Analysis for Microarrays (PAM) software in R revealed a 50-gene signature predictive of the response. After 4 cycles of treatment, a unique geneset encompassing B progenitor markers (B cell response) or T lymphocyte signaling (T cell response) was up-regulated in responding patients, suggesting that induction of immune cell markers reflected a different sensitivity to lenalidomide treatment.To obtain further insights on the modulation of microenvironment by lenalidomide, we took advantage of the transcriptome-based computational method called Microenvironment Cell Populations (MCP)-counter in R package to estimate the abundance of immune (T cells, CD8 T cells, cytotoxic lymphocytes, NK cells, B cells, monocytes, myeloid dendritic cells) and non-immune stromal (neutrophils, endothelial cells and fibroblasts) cell populations from RNA-sequencing data. Compared to controls, the relative abundance of T, cytotoxic, B, NK lymphocytes and myeloid dendritic cells was significantly lower in patients irrespective of the response to lenalidomide. After 4 cycles of treatment, the abundance of cytotoxic T cells significantly increased while other populations NK cells, monocytes, neutrophils, myeloid dendritic cells, endothelial cells and fibroblasts did not vary significantly. Patients with a T cell response under therapy had less abundant monocyte, and trend to less abundant B cell and endothelial cells compared to those with a B cell response. Consistent with the induction of a T cell response, IFNγ level increased significantly after treatment in responders. Altogether, these analyses demonstrate that immune cell populations including cytotoxic T cells are recruited as potential effectors of immune response upon lenalidomide.Conclusion: Our results show that lenalidomide induces an adaptive immune response that may participate in the erythroid improvement in non-del(5q) low/int-1 risk MDS. Future investigations will help understanding the architecture of immune cells and inflammation in the bone marrow microenvironment that may influence the prognosis. DisclosuresFenaux:Amgen: Honoraria, Research Funding; Astex: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Astex: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Fontenay:Celgene: Research Funding.

Successful Treatment of Concurrently Diagnosed Multiple Myeloma and Myelodysplastic Syndrome with Isolated Del(5q) with Lenalidomide, Bortezomib, and Dexamethasone

Lenalidomide is known to be an effective therapy for multiple myeloma (MM) and for myelodysplastic syndrome with isolated del(5q). However, there have been very few reports of treatment of both conditions using lenalidomide when they are diagnosed concurrently. A review of the literature revealed two reports of MM and del(5q) MDS treated with lenalidomide. We report the case of a patient simultaneously diagnosed with multiple myeloma and myelodysplastic syndrome with isolated del(5q) who was treated successfully with lenalidomide. The patient is a 74 year old female who was referred to hematology for worsening chronic macrocytic anemia with a hemoglobin of 9.4 g/dL. A serum protein electrophoresis (SPEP) was obtained during her workup and demonstrated an IgG kappa monoclonal spike of 4.7 g/dL. Free light chain analysis demonstrated a kappa/lambda ratio of 36.7. The patient was mildly hypercalcemic at 10.6 g/dL but had no renal insufficiency. Platelet and white blood cell counts were normal. There were no osteolytic lesions on skeletal survey and a whole body PET scan identified no bony disease or plasmacytomas. A β-2 microglobulin level was 3.7 mg/L and albumin was 3.3 g/dL. Bone marrow biopsy revealed 60% plasma cells in a 70% cellular marrow. Granulocytic and megakaryocytic dysplasia was identified. Fluorescence in situ hybridization returned showing a 4:14 translocation in 72% of analyzed nuclei and monosomy 13 in 61% of nuclei analyzed consistent with an unfavorable risk profile. Chromosome analysis also revealed a 5q deletion in 15 of 20 analyzed cells. Bone marrow blasts were measured at 1%. Therefore, the patient concurrently met diagnostic criteria for stage II IgG kappa multiple myeloma per the International Staging System and low risk myelodysplastic syndrome with isolated del(5q) per the 2016 WHO classification of MDS with a Revised International Prognostic Scoring System Score (IPSS-R) of 2. She was started on lenalidomide 25 mg daily, bortezomib 1.3 mg/m2 on days 1, 4, 8, and 11 and dexamethasone 20 mg on days 1, 8, and 15 of a 21 day cycle. After 3 cycles of therapy, serum immunofixation electrophoresis showed an unquantifiably low IgG kappa monoclonal spike and the patient's kappa/gamma light chain ratio had normalized to 1.1. Hemoglobin and calcium returned to normal. On repeat bone marrow biopsy, there was normocellular marrow with 4% polytypic plasma cells by kappa/lambda immunohistochemistry. No dysplasia was identified and bone marrow blasts were 1.5%. Therefore, the patient achieved a very good partial response (VGPR) to therapy for multiple myeloma according to International Myeloma Working Group criteria within 3 months. She met National Comprehensive Cancer Network criteria for response of her MDS to lenalidomide by normalization of hemoglobin. The patient's case demonstrates successful treatment of concurrently diagnosed multiple myeloma and MDS with isolated del(5q) using lenalidomide. Among the two other similar cases we discovered in the literature, one patient was treated with low-dose lenalidomide and dexamethasone [Nolte, et al. Eur J Haematol. 2017 Mar;98(3):302-310.], and the other patient was treated with high-dose lenalidomide and dexamethasone, achieving a partial response [Ortega, et al. Leuk Res. 2013 Oct;37(10):1248-50.]. Neither patient received a proteasome inhibitor. In our case, the patient was treated with higher intensity induction therapy for multiple myeloma and achieved a VGPR. She did not have worsening cytopenias during therapy, and in fact experienced normalization of her blood counts. Therefore, it is reasonable to treat patients simultaneously diagnosed with MM and MDS with isolated del(5q) with standard three-drug induction therapy for multiple myeloma. While our approach makes sense in the abstract, hematology/oncologists should be aware that it works in practice. Disclosures No relevant conflicts of interest to declare.

Identification of Lenalidomide Sensitivity and Resistance Mechanisms in Non-Del(5q) Myelodysplastic Syndromes.

Whereas lenalidomide is an effective therapy for del(5q) MDS patients, a minority of non-del(5q) MDS patients achieve hematologic improvement with lenalidomide. We used computational biology modeling and digital drug simulation to examine genomic data from 56 non-del(5q) MDS patients treated with lenalidomide, and then matched treatment response with molecular pathways. The computer inferred genomic abnormalities associating with lenalidomide treatment response in non-del(5q) MDS to include trisomy 8, del(20q), or RUNX1 loss of function mutations. Genomic abnormalities associating with lenalidomide resistance in non-del(5q) MDS patients included mutations in SF3B1, TET2, WNT3A amplification, MCL1 amplification, and/or PSEN2 amplification. These results may inform protocols for determining appropriateness of lenalidomide in non-del(5q) MDS.

Resistance to Lenalidomide in Del(5q) MDS Is Mediated By Inhibition of Drug-Induced Megakaryocytic Differentiation

The immunomodulatory drug lenalidomide (LEN) is the treatment of choice for del(5q) MDS patients. LEN has been shown to trigger the specific degradation of CSNK1A1 and IKZF1 proteins after binding the E3-ligase substrate adaptor CRBN. When brought below a certain expression threshold, CSNK1A1 deficiency activates a p53-dependent apoptotic response. Thus, the unique sensitivity of del(5q) cells to LEN is explained by CSNK1A1 haploinsufficiency in del(5q) MDS patients. Despite its efficacy, 50% of LEN-treated patients eventually relapse within an interval of 2-3 years after treatment. Treatment failure is associated to low platelet counts and occurrence of additional mutations, such as TP53. To identify novel genetic determinants of LEN resistance, we have compared whole genome sequencing data of paired samples from six del(5q) patients who have been treated with LEN and eventually became resistant to the treatment. We identified 2 patients with mutations in TP53. The remaining four presented RUNX1 alterations: two patients had protein coding mutations in RUNX1 and two had a significant reduction in RUNX1, but not TP53, transcript levels.As a model of sensitivity, we studied the response to LEN in two human del(5q) cell lines, MDS-L and KG-1a. RUNX1 protein levels are postranscriptionally upregulated upon exposure to LEN, accompanied by increased levels of RUNX1 activity. Deletion of CRBN expression cancelled these effects. RUNX1 overexpression inhibited clonogenic growth and induced apoptosis. We then generated RUNX1 knock-out (KO) clones derived from MDS-L cells using CRISPR/Cas9 system. RUNX1 KO cells presented increased proliferation, increased colony growth and reduced apoptosis in the presence of LEN compared to wild-type (WT) control clones. These results were validated with different shRNAs against RUNX1. Genetic rescue experiments showed that RUNX1 mutants were unable to restore sensitivity to the drug compared to RUNX1 WT. Finally, modeling RUNX1 loss-of-function (LOF) in CSNK1A-depleted human CD34+ cells abrogated the effects of LEN on colony forming cell assays. Thus, RUNX1 function is required for the elimination of del(5q) cells by LEN.To understand the molecular mechanisms underlying the resistant phenotype, we performed RNA-seq on MDS-L cells treated with LEN for 24h. We observed a significant upregulation of Platelet specific genes (ITGB3, ITGA2B, VWF, THBD, SELP, TREML1, GATA1) coupled to downregulation of Cell Cycle genes (E2F2, E2F1, MCM5, CDKN1A), suggesting that LEN induces differentiation in to the Megakaryocytic (Meg) lineage. We found a significant upregulation of CD41+/CD61+ double positive cells after LEN exposure in vitro and in vivo, associated to the appearance of multinucleated cells. Importantly, the apoptotic response was associated to the emergence of the differentiating population. At the molecular level, CRBN is required for LEN-induced differentiation. Further downstream we identified IKZF1 degradation as key trigger, as IKZF1 overexpression restrained Meg differentiation and a IKZF1 dominant negative isoform enhanced it. In contrast, CSNK1A overexpression did not alter differentiation after LEN, but did reduce apoptotic induction. Moreover, we identified GATA2 targets enriched in LEN-regulated genes and showed that GATA2 overexpression or downregulation using shRNAs significantly increased or reduced LEN induced differentiation respectively.Finally, gene expression analysis after LEN exposure showed that Meg signatures were not enriched in resistant RUNX1 KO cells compared to WT control. Accordingly, RUNX1 KO cells did not undergo differentiation upon LEN exposure. RUNX1 LOF in CSNK1A-depleted primary human CD34+ cells blocked CFU-Mk growth in LEN treated cells. GATA2 overexpression was unable to restore LEN effects in RUNX1 deficient cells, suggesting a cooperative mechanism between both transcription factors. Luciferase assays using the human CD41 promoter showed that RUNX1 mutants reduced promoter transactivation compared to RUNX1 WT. Remarkably, we observed a similar phenotype for LEN-resistant TP53 KO cells.As a conclusion, our results suggest that GATA2, RUNX1 and TP53 cooperate to drive Meg differentiation after LEN-mediated degradation of IKZF1 protein. Loss of function mutations affecting RUNX1 or TP53 alter the activity of GATA2 transcriptional complex, rendering del(5q) cells unresponsive to LEN. DisclosuresPlatzbecker:Celgene: Research Funding.

Targeted Delivery of CpG-Mir-146a Mimic Oligonucleotides As a Therapeutic Strategy to Reduce NF-Κb-Mediated Pathogenic Inflammation and Myeloid Leukemia Progression

NF-kB signaling plays central role in the regulation of immune cell activity. The microRNA-146a-5p (miR-146a) provides negative feedback inhibition of the NF-kB pathway to prevent either excessive immunity, such as cytokine release syndrome. Low expression of miR-146a is also implicated in certain types of leukemia, especially in del(5q)-syndrome myelodysplastic and acute myeloid leukemia (MDS/AML). While miR-146a is a potential therapeutic target, the lack of efficient miRNA delivery methods limits clinical translation. We previously developed a strategy for targeted delivery of oligonucleotide therapeutics, such as siRNA, into human and mouse myeloid cells and B cells, using partly or completely phophorothioated (PS) single-stranded oligodeoxynucleotides containing CpG motif (CpG ODN). Here, we demonstrate that similar strategy, using CpG ODN optimized for targeting human monocytes, can be employed for the delivery of functional miR-146a mimic. The CpG-miR146a mimic conjugate, but not unconjugated miR-146a, was quickly internalized by target human and mouse myeloid cells, such as dendritic cells, macrophages and leukemic cells. In vitro CpG-miR146a mimic reduced protein levels of downstream targets, Irak1 and Traf6, as well as the inhibition of DNA binding and transcriptional activity of NF-kB in myeloid cells. We further verified functional activity of CpG-miR146a mimic against several models of acute inflammation. The intravenous injections of this oligonucleotide reduced myeloproliferation and excessive cytokine response to bacterial endotoxin or to live Listeria challenge in miR-146a-/- mice. Furthermore, CpG-miR146a mimic had similar anti-inflammatory effect also in a human model of cytokine release syndrome induced by CD19 chimeric antigen receptor (CAR) T-cell activity. CpG-miR146a effectively abrogated CAR T-cell induced IL-6 production by human monocytes in vitro. Finally, our studies indicated that CpG-miR146a mimic can target del(5q) MDS and AML cells in vitro and in vivo resulting in growth inhibition of MDSL and HL-60 acute myeloid leukemia. Our results suggest that CpG-miR146a strategy can be provide a new-in-class immunomodulatory therapeutics for treatment of acute inflammatory disorders, myeloproliferative diseases and myeloid leukemia. DisclosuresWang:Mustang Therapeutics: Other: Licensing Agreement, Patents & Royalties, Research Funding. Forman:Mustang Therapeutics: Other: Licensing Agreement, Patents & Royalties, Research Funding. Kortylewski:N/A: Patents & Royalties: I am an inventor on the US patent 9,976,147.

Importance of Transcription Factor Nrf2 for Cereblon Expression and Clinical Response to Combination of Lenalidomide and Erythropoietin in Lower-Risk Myelodysplastic Syndromes

INTRODUCTIONNrf2 (nuclear factor, erythroid-derived 2-like 2 or NF-E2-related factor 2) is a transcription factor involved in antioxidant response by reducing oxidative stress. Erythropoietin (EPO) was described as an inducer of Nrf2 in the brain. Nrf2 binds to the promoter of gene coding for cereblon (CRBN) and stimulates CRBN expression (Lee et al. Biochem.Biphys Res Commun 2010; 399: 711-715). We showed that the high level of full length CRBN mRNA and CRBN protein is important for the efficacy of lenalidomide (LEN) in lower-risk MDS patients (Jonasova et al. Eur J Haematol 2015; 95: 27-34; Fuchs et al. Leuk Res 2017; 55 S1: S132, abstr. 227). Addition of EPO to LEN restored transfusion independence of MDS patients when their anemia relapsed during the course of LEN treatment (Jonasova et al. Leuk Res 2018; 69: 12-17). LEN inhibits E3 ubiquitin ligase RNF41 (ring finger protein 41), which polyubiquitinates EPO receptor (EPOR) and marks it for degradation in proteasomes. (Basiorka et al. Cancer Res 2016; 76: 3531-3540).AIMSThe aim of our study was to evaluate the levels of Nrf2 mRNA and CRBN mRNA in mononuclear cells obtained from peripheral blood of lower-risk MDS patients after addition of EPO to lenalidomide in MDS patients who relapsed during LEN monotherapy. The further goal was to study the effect of LEN, EPO and combination of LEN plus EPO in cell lines (SKM-1 leukemic cell line established from a patient with progression of MDS to myelomonocytic leukemia and in MDS-L cells a human myeloblastic cell line, established as a MDS92 subline from the bone marrow of an MDS patient with del(5q) (Matsuoka et al. Leukemia 2010; 24: 748-755).METHODSNrf2 mRNA and full-length CRBN mRNA levels were quantified in 9 lower-risk MDS patients with del(5q) and in 2 MDS patients with normal chromosome 5 [nondel(5q)] who were previously resistant to EPO treatment. All patients with median hemoglobin level 80 g/l were transfusion dependent before starting the therapy with LEN (5 or 10 mg/day). Recombinant human EPO (rHuEPO) in dose of 40,000 IU s.c. per week was combined with LEN in these MDS patients who relapsed during LEN monotherapy or were resistant to LEN [one non(del5q) patient]. Four lower-risk MDS patients responsive to EPO after diagnosis were also used for comparison of Nrf2 mRNA and full-length CRBN mRNA levels. Mononuclear cells were isolated by Ficoll-Paque PLUS gradient separation, then washed by phosphate buffered saline and remaining red cells were lysed. Both SKM-1 and MDS-L cells were incubated without or with LEN, EPO and combination of LEN plus EPO for 19, 24 and 43 hours. The end concentration of added Epo was 2U/ml and LEN 10 µM. The levels of Nrf2 mRNA and full length CRBN mRNA were measured using the reverse transcription-quantitative TaqMan PCR assay.RESULTSThe levels of Nrf2 mRNA and full-length CRBN mRNA in peripheral blood mononuclear cells correspond during rHuEPO monotherapy of four responsive lower-risk MDS patients. In these cases no increase of Nrf2 mRNA and CRBN mRNA levels was found. Increased Nrf2 mRNA and CRBN mRNA levels in peripheral blood mononuclear cells were found after addition of rHuEPO to lenalidomide in six MDS patients with del(5q) and one non(del5q) MDS patient who relapsed during LEN monotherapy. All these patients responded to combination of rHuEPO and lenalidomide by increased hemoglobin levels. Addition of or rHuEPO to lenalidomide was without effect on Nrf2 mRNA and CRBN mRNA levels in one non(del5q) patient who was resistant to LEN. Experiments with SKM-1 and MDS-L cells showed that rHuEPO alone did not increased Nrf2 mRNA and CRBN mRNA levels. However LEN and in a greater extent the combination of rHuEPO and LEN increased Nrf2 mRNA and CRBN mRNA levels.CONCLUSIONSOur findings indicate that transcription factor Nrf2 is involved in CRBN expression in mononuclear cells obtained from peripheral blood of lower-risk MDS patients and in SKM-1 and MDS-L cells in culture. Expression of both Nrf2 and CRBN is stimulated by lenalidomide and in a greater extent by combination of lenalidomide and rHuEPO. Measurement of CRBN mRNA level as an important factor for prediction of the efficacy of not only lenalidomide monotherapy but also of combination of LEN and rHuEPO.This work was supported by the research project for conceptual development of research organization (00023736; Institute of Hematology and Blood Transfusion, Prague) and Grant Agency of Charles University, project number 924616. DisclosuresNo relevant conflicts of interest to declare.

HSPA9 Gene Regulates Erythroid Maturation in Human Hematopoietic Progenitor cells

Background and HypothesisMyelodysplastic syndromes (MDS) is the most common form of adult myeloid blood cancers within the United States. A high fraction of patients with MDS can further progress to acute myeloid leukemia (AML) without intervention. MDS is a heterogeneous group of blood cancers characterized by abnormal hematopoietic cell maturation, increased apoptosis of bone marrow cells, and peripheral blood cytopenias. Low blood cell counts in MDS patients result in morbidity and mortality due to dysregulation of hemostasis and infection. Up to 25% of MDS patients acquire an interstitial deletion on chromosome 5q, also known as Del(5q). The HSPA9 gene is located in the commonly deleted region and is associated with patients that progress from MDS to AML.We hypothesize that that inhibition of the Del(5q) candidate HSPA9 gene can disrupt the erythroid maturation, contributing to the peripheral blood cytopenia observed in MDS patients.Results Knockdown of HSPA9 inhibited cell growth, increased apoptosis, inhibited erythroid maturation, and increased TP53 levels in human CD34+ cells. In order to test the effects of HSPA9 knockdown in human CD34+, we first utilized the siRNA that specifically targets the HSPA9 gene. HSPA9 specific siRNA reduced the HSPA9 protein expression level to approximately 50% compared to control siRNA. Knockdown of HSPA9 by siRNA inhibited cell growth, increased apoptosis, inhibited erythroid maturation, and increased TP53 levels in human CD34+ cells. Pharmacologic inhibition of HSPA9 induced apoptosis, decreased erythroid maturation and increased TP53 levels in human CD34+ cells. MKT‐077 is an HSPA9 inhibitor and it reduced HSPA9 expression, inhibited cell growth, increased apoptosis, inhibited erythroid maturation and induced TP53 expression in a dose‐dependent manner in human CD34+ cells. Reduction of TP53 reversed the inhibition of erythroid maturation caused by HSPA9 knockdown. We constructed two TP53 shRNAs which were able to decrease TP53 levels by about 80% and 50%, respectively, compared to control shRNA. We co‐transduced lentiviruses containing shRNA targeting HSPA9 and TP53 into human CD34+ cells using double antibiotic selection. The erythroid inhibition following HSPA9 knockdown was significantly decreased and reversed following simultaneous knockdown of TP53 with shTP53. ConclusionPrevious studies showed that Del(5q) MDS bone marrow samples display an induction of TP53 in erythroid cells and upregulation of TP53 target gene expression. Our study focuses on the effects of HSPA9 on erythroid maturation, as well as cell growth and apoptosis in human CD34+ hematopoietic progenitor cells. We observed that inhibition of HSPA9 (by siRNA, short hairpin RNA and MKT‐077), a gene on the proximal CDR of chromosome 5, caused cell growth inhibition, apoptosis induction and erythroid maturation inhibition in human CD34+ cells. These phenotypes were associated with increased TP53 levels. We also found that inhibition of TP53 in human CD34+ cells could reverse erythroid inhibition caused by HSPA9 inhibition. In conclusion, our study indicates that inhibition of HSPA9 gene can inhibit erythroid maturation, which may contribute to the peripheral blood cytopenia seen in Del(5q) MDS patients. Our data also suggests the inhibition of erythroid maturation via HSPA9 inhibition may be regulated through a TP53‐dependent mechanism.Support or Funding InformationThis work is supported by West Virginia School of Osteopathic Medicine Faculty Startup Funding (T. Liu).

Megakaryocytes harbour the del(5q) abnormality despite complete clinical and cytogenetic remission induced by lenalidomide treatment.

The mechanisms underlying lenalidomide-resistance of del(5q) MDS stem cells remain to be elucidated and may include cell-intrinsic as well as microenvironmental causes. Abnormal hypolobated megakaryocytes constitute one of the hallmarks of del(5q) MDS. We hypothesized that these cells have potential implications for the regulation of haematopoietic stem cells (HSC) similarly to what has recently been described for megakaryocytes in the murine system. Therefore, we conducted a study to determine the response of abnormal hypolobated megakaryocytes to lenalidomide therapy. We studied lenalidomide-treated patients in the MDS-004 trial as well as a cohort seen at our institution. Morphological evaluation at time of complete cytogenetic remission (CCyR) demonstrated the persistence of hypolobated megakaryocytes in all evaluable patients (n=9). Furthermore, we provide evidence that the abnormal hypolobated morphology is restricted to del(5q) megakaryocytes, both at diagnosis and during CCyR. Using fluorescence insitu hybridisation analysis on flow-sorted stem- and progenitor populations, we observed a similar degree of clonal involvement in megakaryocyte-erythroid-progenitors as in HSC. Taken together, our findings suggest that megakaryocyte morphology might aid in the evaluation of patients where discontinuation of lenalidomide is considered and offers interesting hypotheses for further investigation of lenalidomide resistance.

Delayed Globin Synthesis Leads to Excessive Heme and the Macrocytic Anemia of Diamond Blackfan Anemia and del(5q) Myelodysplastic Syndrome

Thirty percent of Diamond Blackfan anemia (DBA) cases result from haploinsufficiency of ribosomal protein S19 and ~40% from haploinsufficiencies of 15 other ribosomal proteins. The macrocytic anemia of myelodysplasia with deletion of chromosome 5q (del(5q)MDS), which results from the acquisition of RPS14 haploinsufficiency, has a similar clinical phenotype. Although these mutations disrupt ribosome assembly and impair protein translation, how this causes macrocytic anemia remains uncertain and controversial. Since 95% of the protein content of red cells is globin, we hypothesized that any germline or somatic mutation that slows protein synthesis would impair globin production relative to heme production. This is because the synthesis of heme, a chemical chelate, depends on only small amounts of protein (enzymes) and the rate limiting enzyme, ALAS2, is an early GATA1 target. Studies of marrow cells from patients with DBA and del(5q) MDS show that heme synthesis indeed progresses normally, while globin synthesis is delayed. This results in excess heme in CFU-E/proerythroblasts, excessive ROS and cell death (Sci Transl Med 8:338RA67, 2016). Similar results are seen in a murine model of heme excess (Flvcr1 -deletion) (J Clin Invest 125:4681, 2015) and murine models of DBA. As slowing heme synthesis improves the coordination of heme and globin and improves red cell production, a phase 2 study is underway (PI Bart Scott) to determine the efficacy of aggressive iron chelation to slow heme synthesis in patients with very low to intermediate risk MDS and anemia. More recently, we have quantitated the cell surface expression of CD71, CD44 and Ter119 on individual murine erythroid cells from normal, Flvcr1 -deleted mice with macrocytic anemia, and erythropoietin-treated mice. We then barcoded and assessed the cell's total transcriptome. By linking these datasets, we uncovered a GATA1-heme autoregulatory loop which regulates normal erythropoiesis and contributes to the failed erythropoiesis of ribosomal protein haploinsufficiency. We show that heme normally upregulates ribosome protein transcription in early erythroid cells. Thus, in addition to increasing globin transcription and translation (via Bach1 and HRI), heme assures adequate ribosomes for globin synthesis. In later erythroid cells, heme decreases GATA1, GATA1 target genes and mitotic spindle gene expression, assuring that red cell differentiation appropriately terminates and cell division ceases. In human marrow CD36+GlyA- or CD36+GlyA+ cells, these changes occur within 15 minutes of inducing endogenous heme synthesis with ALA (bypasses ALAS2) and iron. As excess heme would increase ROS, increase ribosomal protein imbalance to intensify P53 expression, prematurely lower GATA1, and impede mitosis, our data explain the ineffective (early termination of) erythropoiesis in DBA and del(5q) MDS, help explain why these anemias are macrocytic, and reconcile the disparate observations of others. DisclosuresNo relevant conflicts of interest to declare.

Cereblon and Redox in Plasma Cells

The protein Cereblon (CRBN) has recently attracted considerable interest by virtue of its ability to mediate both anti-tumor as well as teratogenic activities of immunomodulatory drugs (IMiDs) 1-3 . Certain IMiDs have been shown to re-direct CRBN-associated ubiquitylation activity towards neo-substrates such as IKZF1/3 and CK1a, thereby promoting their proteasomal degradation 4-8. This mechanism has been implicated in the anti-proliferative effects of IMiDs in multiple myeloma (MM) and del(5q)-MDS. However, the pleiotropic anti-tumor effects of IMiDs, their adverse effects such as teratogenicity and neurotoxicity, and their synergy with proteasome inhibitors suggests further mechanisms of action. Previous research on CRBN has revealed novel insights that shed light on the physiological functions of this versatile protein in plasma cells. Indeed, next to its function as a ubiquitin ligase, CRBN has been shown to exist in ubiquitin-ligase independent complexes, where it functions as a chaperone for crucial trans-membrane receptors such as CD147 (also known as Basigin) and MCT1 (monocarboxylate transporter 1) 9. This functions links CRBN to the regulation of angiogenesis, cell invasion and particularly cell metabolism by governing lactate homeostasis. Likewise, CRBN has been implicated in the regulation of TrxR (thioredoxin reductase), thereby critically determining the redox balance in plasma cells 10. These findings have important implications for our understanding of IMiD biology and for the development of novel treatment approaches and predictive biomarkers for MM. Indeed, IMiDs inhibit the chaperone function of CRBN in a competitive manner and attenuate TrxR via CRBN, thereby preventing intracellular H2O2 decomposition. In turn, CRBN-dependent degradation of IKZF1 and IKZF3 have been shown to be the result high H2O2 levels. Against this background, CRBN-regulated proteins such as CD147 and MCT1 as well as TrxR may serve as new therapeutic target structures while functional read-outs as CD147 and MCT1 destabilization or antioxidative capacity may serve as predictive biomarkers for IMiD response.1. Ito T, Ando H, Suzuki T, et al. Identification of a primary target of thalidomide teratogenicity. Science . 2010;327(5971):1345- 1350.2. Zhu YX, Braggio E, Shi CX, et al. Cereblon expression is required for the antimyeloma activity of lenalidomide and pomalidomide. Blood . 2011;118(18):4771-4779.3. Lopez-Girona A, Mendy D, Ito T, et al. Cereblon is a direct protein target for immunomodulatory and antiproliferative activities of lenalidomide and pomalidomide. Leukemia . 2012;26(11):2326-2335.4. Kronke J, Udeshi ND, Narla A, et al. Lenalidomide causes selective degradation of IKZF1 and IKZF3 in multiple myeloma cells. Science . 2014;343(6168):301-305.5. Lu G, Middleton RE, Sun H, et al. The myeloma drug lenalidomide promotes the cereblon-dependent destruction of Ikaros proteins. Science . 2014;343(6168):305-309.6. Gandhi AK, Kang J, Havens CG, et al. Immunomodulatory agents lenalidomide and pomalidomide co-stimulate T cells by inducing degradation of T cell repressors Ikaros and Aiolos via modulation of the E3 ubiquitin ligase complex CRL4(CRBN.). Br J Haematol . 2014;164(6):811-821.7. Zhu YX, Braggio E, Shi CX, et al. Identification of cereblon-binding proteins and relationship with response and survival after IMiDs in multiple myeloma. Blood . 2014;124(4):536-545.8. Kronke J, Fink EC, Hollenbach PW, et al. Lenalidomide induces ubiquitination and degradation of CK1alpha in del(5q) MDS. Nature . 2015;523(7559):183-188.9. Eichner R, Heider M, Fernandez-Saiz V, et al. Immunomodulatory drugs disrupt the cereblon-CD147-MCT1 axis to exert antitumor activity and teratogenicity. Nat Med . 2016;22(7):735-743.10. Sebastian S, Zhu YX, Braggio E, et al. Multiple myeloma cells' capacity to decompose H2O2 determines lenalidomide sensitivity. Blood . 2017;129(8):991-1007. DisclosuresBassermann:Celgene: Research Funding.

Survival of Patients with Del(5q) Syndrome in the Lenalidomide Era

▪Background: Myelodysplastic syndrome with deletion 5q [del(5q) MDS] is characterized by higher incidence in older women, progressive anemia, and >70% rate of hematologic response to single-agent lenalidomide (Len). Len was approved for del(5q) MDS by the United States Food and Drug Administration (FDA) in 2005 on the basis of response rates, with uncertain effect on overall survival (OS). Our objective was to evaluate whether the availability of Len affected the frequency of del(5q) designation among newly diagnosed MDS cases in the US, and to compare OS of del(5q) MDS patients treated with upfront Len or with hypomethylating agents (HMA, azacitidine or decitabine), which were an alternative therapy for MDS available in the US.Methods: We selected cases of low-grade MDS diagnosed in 2001-2011 from the linked Surveillance, Epidemiology, and End Results-Medicare (SEER-Medicare) data base. Patients were >65 years of age at diagnosis. We identified treatment with Len or HMA in the subpopulation of del(5q) cases diagnosed in 2007-2011 who had complete records of injectable and oral chemotherapy drugs (i.e. with Medicare Part D coverage). Transfusion dependence was assessed by identifying all transfusions within 3 months before therapy, and in months 1-2, 3-4, or 5-6 after treatment initiation. We compared overall survival (OS) using log-rank tests, and in multivariable Cox models adjusting for available confounders, reporting adjusted hazard ratios (HR) with 95% confidence intervals (CI).Results: Among 21,384 low-grade MDS patients, the frequency of del(5q) MDS designation (N=655) doubled after approval of Len (2% in 2001-06 vs. 4% in 2007-11, P<.0001). Among del(5q) MDS patients with complete Medicare claims, those diagnosed in 2007-11 were slightly less likely to require transfusion at diagnosis compared with 2001-06 cases (73% vs. 82%, P=.056), and significantly more likely to receive chemotherapy (52% vs. 37%, P<.0001). There was no significant change in OS (from diagnosis) among del(5q) MDS patients between the two periods (P=.27), even after adjustment for age, socioeconomic factors, and baseline intensity of transfusions (adjusted HR, 0.96, CI, 0.77-1.19, P=.70). Median OS from diagnosis in del(5q) MDS was 2.3 years (CI, 2.0-2.6), not significantly different from other low-grade MDS (2.2 years, CI, 2.2-2.3, P=.09). OS at 3-years was 41% for del(5q) vs. 42% for other low-grade MDS.We compared outcomes of del(5q) MDS patients from 2007-2011 (N=157) who had complete record of oral prescriptions (i.e. enrollees in a Medicare Part D plan), including 114 patients treated with upfront Len and 43 treated with upfront HMA. Patients treated with Len were on average older (median age 81 years for Len vs. 78 for HMA, P=.007), more often female (75% vs. 51%, P=.007), with poor functional status (16% vs. 2%, P=.026) or with Medicaid co-insurance (24% vs. 9%, P=.046). There was no significant difference in time from diagnosis to treatment (median 3 months, P=.10), comorbidity index (P=.22), prior hospitalization (P=1.0) or prior transfusions (P=1.00). However, HMA-treated cases had a higher prevalence of pre-treatment G-CSF use (21% vs. 4%, P=.003) and more frequently a subsequent record of acute leukemia (12% vs. 2%, P=.017). OS was significantly better among patients treated with Len than with HMA (median 2.8 vs. 0.9 years, Fig. A), and remained so in a multivariable model adjusting for age, sex, race, baseline transfusion dependence, prior G-CSF, hospitalization, and poor functional status (adjusted HR, 0.27, CI, 0.16-0.43, P<.0001). Len was also associated with a significantly higher rate of transfusion independence within 6 months of therapy initiation (68% vs. 30%, P=.00002, Fig. B).Conclusions:In this first population-based assessment of survival outcomes among US patients with del(5q) MDS, the FDA approval of Len led to an increased designation of del(5q) among MDS cases, which will confound studies relying on historical cohort comparisons. Rates of transfusion independence after Len in del(5q) MDS were similar to experience from clinical trials. A significant proportion of del(5q) MDS patients do not receive Len, and state-subsidized co-insurance was associated with improved access to this expensive drug. Among del(5q) MDS patients who receive active therapy, upfront Len is associated with a significantly better OS and a higher rate of transfusion independence than upfront HMA. [Display omitted] DisclosuresReagan:Seattle Genetics: Membership on an entity's Board of Directors or advisory committees; Alexion: Honoraria. Olszewski:TG Therapeutics: Research Funding; Genentech: Research Funding; Bristol-Myers Squibb: Consultancy.