Pathophysiology Of Myelodysplastic Syndromes Research Articles

Genetic iron overload aggravates, and pharmacological iron restriction improves, MDS pathophysiology in a preclinical study

AbstractAlthough iron overload is a common feature in myelodysplastic syndromes (MDSs), it remains unclear how iron excess is detrimental for disease pathophysiology. Taking advantage of complementary approaches, we analyzed the impact of iron overload and restriction achieved through genetic activation of ferroportin (FPN) via the C326S mutation (FPNC326S) and pharmacologic inhibition (vamifeport) of the iron exporter FPN, respectively, in a MDS mouse model. Although FPNC326S-induced iron overload did not significantly improve the late stages of erythroid maturation, vamifeport-mediated iron restriction ameliorated anemia and red blood cell maturation in MDS mice, through the reduction of oxidative stress and apoptosis in erythroid progenitors. Iron overload aggravated, and restriction alleviated, reactive oxygen species formation, DNA damage, and cell death in hematopoietic stem and progenitor cells, resulting in altered cell survival and quality. Finally, myeloid bias, indicated by expanded bone marrow myeloid progenitors and circulating immature myeloid blasts, was exacerbated by iron excess and attenuated by iron restriction. Overall, vamifeport treatment resulted in improved anemia and significant survival increment in MDS mice. Interestingly, the combined therapy with vamifeport and the erythroid maturation agent luspatercept has superior effect in improving anemia and myeloid bias as compared with single treatments and offers additive beneficial effects in MDS. Our results prove, to our knowledge, for the first time in a preclinical model, that iron plays a pathologic role in transfusion-independent MDS. This is likely aggravated by transfusional iron overload, as suggested by observations in the FPNC326SMDS model. Ultimately, the beneficial effects of pharmacologic FPN inhibition uncovers the therapeutic potential of early prevention of iron toxicity in transfusion-independent MDS.

RNA splicing as a therapeutic target in myelodysplastic syndromes

Myelodysplastic syndromes (MDS) represent a heterogeneous group of hematological disorders and are more commonly found in people over the age of 60. MDS patients exhibit peripheral blood cytopenias and carry an increased risk of disease progression to acute myeloid leukemia (AML). Splicing factor mutations (including genes SF3B1, SRSF2, U2AF1 and ZRSR2) are early events identified in more than 50% of MDS cases. These mutations cause aberrant pre-mRNA splicing and impact MDS pathophysiology. Emerging evidence shows that splicing factor-mutant cells are more sensitive to perturbations targeting the spliceosome, aberrantly spliced genes and/or their regulated molecular pathways. This review summarizes current therapeutic strategies and ongoing efforts targeting splicing factor mutations for the treatment of MDS.

Modelling and drug targeting of a myeloid neoplasm with atypical 3q26/MECOM rearrangement using patient-specific iPSCs.

Structural variations involving enhancer hijacking induce aberrant oncogene expression and cause tumorigenesis. A rare translocation, t(3;8)(q26.2;q24), is associated with MECOM and MYC rearrangement, causing myeloid neoplasms with a dismal prognosis. The most recent World Health Organization classification recognises myeloid neoplasms with MECOM rearrangement as acute myeloid leukaemia (AML) with defining genetic abnormalities. Recently, the increasing use of induced pluripotent stem cell (iPSC) technology has helped elucidate the pathogenic processes of haematological malignancies. However, its utility for investigating enhancer hijacking in myeloid neoplasms remains unclear. In this study, we generated iPSC lines from patients with myelodysplastic syndromes (MDS) harbouring t(3;8)(q26.2;q24) and differentiated them into haematopoietic progenitor cells to model the pathophysiology of MDS with t(3;8)(q26.2;q24). Our iPSC model reproduced the primary patient's MECOM expression changes and histone H3 lysine 27 acetylation (H3K27ac) patterns in the MECOM promoter and MYC blood enhancer cluster (BENC). Furthermore, we revealed the apoptotic effects of the bromodomain and extra-terminal motif (BET) inhibitor on iPSC-derived MDS cells by suppressing activated MECOM. Our study demonstrates the usefulness of iPSC models for uncovering the precise mechanism of enhancer hijacking due to chromosomal structural changes and discovering potential therapeutic drug candidates for cancer treatment.

Risk Factors Associated to Severe Episodes of Infection in Myelodysplastic Syndromes

INTRODUCTION. Mortality differs according to the Revised International Prognostic Scoring System (IPSS-R) risk categories in patients afflicted with myelodysplastic syndromes (MDS). Historically, high-risk (HR)-MDS (IPSS-R >3.5 points) is related to death caused by progression to acute myeloid leukemia (AML), while low-risk (LR)-MDS is associated with mortality due to severe infections (Nachtkamp K, et al. 2016), probably caused by neutrophile dysfunction and hindered oxidative burst. Studies of causes of death (COD) in MDS are scarce. A MD Anderson study in LR-MDS patients identified infections as the leading COD, followed by progression (Dayyani F, et al, 2010), while the Düsseldorf registry recognized infections as the main COD across all MDS categories (Neukirchen, J. et al, 2011). Considering that information in low- and medium-income countries is especially limited, this study aims to describe the characteristics and identify the risk factors for severe infectious events in patients afflicted by MDS in a third-level center in Mexico. METHODS. In this single-center retrospective study, we analyzed severe infection episodes, associated risk factors, and the COD in MDS patients.We included patients diagnosed with MDS from January 2000 to December 2021, according to the 2016 WHO criteria. We registered baseline characteristics and described the episodes of severe infection, defined as hospitalization events due to infectious diseases complicated with septic shock and/or respiratory failure requiring orotracheal intubation. We conducted uni- and multivariate analyses based on the following variables: neutropenia (≤0.5x10 9/L), monocytopenia (≤0.2x10 9/L), lymphopenia (≤1.0x10 9/L), ferritin (≥1,000ng/mL), autoimmune disease, MDS comorbidity index, COPD, granulocyte dysplasia, and treatment with azacytidine or pharmacological immunosuppression. RESULTS. We included 156 patients with MDS treated at our institution. Median age at diagnosis was 65 years (IQR; 55-75 years), 53% of which were women. Multilineage dysplasia was the most prevalent MDS type (59%), followed by excess blasts type 2 (11.5%). LR-MDS represented 68% (n=106), while HR-MDS, 32% (n=50). Hypocellularity in bone marrow biopsy was found in 24.4%. Eighty-seven patients developed 156 infectious episodes, 64.7% of which were severe. A median of 1 serious event (IQR: 0.75-1 event) occurred in each patient, with a median time to diagnosis of 13 months (IQR: 3.5-34.5 months). Infection sites and etiologies are described in Figure 1. Of note, we identified 14 fungal infections, 8 of them by Aspergillus, with a 37.5% death rate. Risk factors associated with increased severe infection risk were neutropenia during follow-up (OR 3.2; 95% CI 1.5-6.4; p=0.001), neutrophil hypogranularity at diagnosis (OR 2.1; CI 95% 1.1-4.1; p=0.029), hyperferritinemia (OR 2.46; CI 95% 1.2-4.9; p=0.011), and azacitidine treatment (OR 3.1; CI 95% 1.01-9.6; p=0.047). Fifty patients (32%) died. In both HR-MDS and LR-MDS groups, the main COD was infection (13 in HR group, 48.1%, vs 10 in LR group, 43.5%; p=0.782), followed by progression to AML (3 in HR group, 11.1%, vs 4 in LR group, 21.7%; p=0.444). Median overall survival was lower in patients with severe neutropenia (62 vs. 210 months; p=0.009) and hyperferritinemia (62 months vs. non-reached, p<0.001) (Figure 2). CONCLUSIONS In our MDS population severe infections represent the leading COD, regardless of risk category. More than half developed at least one infectious episode, 64.7% of which were severe. Fungal infections were highly lethal, suggesting antifungal prophylactic interventions could prove helpful. We found neutropenia during follow-up and hypogranularity at diagnosis to be significant risk factors, the latter perhaps reflecting neutrophil disfunction which should be further evaluated. Hyperferritinemia's association with increased infection and mortality urges to further understand its role in MDS pathophysiology, and could potentially prove a therapeutic target in infections disease prevention. Additionally, there is currently no consensus on infectious disease for use of azacitidine, which might add to a worsening risk of infection.

Combination Therapy with Luspatercept and the Ferroportin Inhibitor Vamifeport Is Superior to Either Drug Alone in Improving Anemia and Reducing Myeloid Skewing in MDS

Background: Patients with myelodysplastic syndromes (MDS) are prone to develop iron overload as a consequence of ineffective erythropoiesis and chronic transfusion therapy. Although iron overload is a common feature in MDS, it has remained unclear whether and how iron excess is detrimental for MDS pathophysiology. Recently, we have shown that iron restriction by the oral ferroportin (FPN) inhibitor vamifeport improves anemia and reduces myeloid skewing, being of benefit for MDS as single treatment. Luspatercept is a recombinant fusion protein acting as TGF-β superfamily ligands trap which has recently gained FDA approval for the treatment of anemia in transfusion-dependent low-risk MDS patients. Luspatercept promotes erythropoietin (EPO)-independent maturation of late-stage erythroid cells, resulting in increased hemoglobin and red blood cells (RBCs) count. However, this drug - due to a lack of improvement in myeloid skewing and AML evolution - is considered a non-disease-modifying therapy. Aims: In this study we investigated the effect of a combined therapy using luspatercept and the FPN inhibitor vamifeport in a preclinical MDS mouse model, with the hypothesis that both drugs could have additive effects with further benefit for the disease. Methods: To this end, we administered the FPN inhibitor vamifeport in NUP98-HOXD13 MDS mice for 3 months (0.5 mg/ml into drinking water), combined with luspatercept for the last 2 months (5 mg/kg twice a week, subcutaneous injection), starting at 3 months of age. Mice receiving the combined treatment were compared to those untreated or receiving single treatments. Results: At steady-state MDS mice develop anemia, neutropenia and lymphopenia, together with an iron overload phenotype, hallmarked by inappropriately low hepcidin levels, elevated serum iron and transferrin saturation, non-transferrin-bound iron (NTBI) formation and tissue iron deposition. Vamifeport administration in MDS mice reduced serum iron and NTBI formation and prevented tissue iron loading, either as a single or combined treatment with luspatercept. Luspatercept and vamifeport, as single-agent treatment, ameliorated anemia and the maturation of bone marrow and splenic RBCs in MDS mice. The combined therapy further improved anemia compared to the treatments with either drugs alone, as suggested by the higher hemoglobin levels, hematocrit and RBC count, and the decreased number of early erythroid precursors, sign of more effective erythropoiesis. Importantly, vamifeport, but not luspatercept, significantly altered myeloid skewing in MDS, revealing a major role of the iron status in myeloid expansion. Myeloid bias, monitored as percentage of CD11b + Gr1 + myeloid cells in the bone marrow was attenuated by iron restriction in vamifeport-treated MDS mice compared to control and luspatercept-treated MDS animals. The combined treatment merkedly reduced myeloid skewing in MDS mice, similarly to the single vamifeport treatment. Furthermore, the number of immature cKit + myeloid blasts in the peripheral blood of MDS animals were decreased by vamifeport alone and in combination with luspatercept, but not by luspatercept alone. Finally, splenomegaly was reduced in MDS mice treated with vamifeport alone or in combination with luspatercept, confirming that iron restriction improves ineffective erythropoiesis and limits myeloid skewing. Overall, the combination therapy with vamifeport and luspatercept showed superior effects in improving anemia and myeloid bias as compared to single treatments. Conclusion: In conclusion, our results show that iron restriction by vamifeport enhances the erythroid maturation action of luspatercept, likely by improving iron utilization in erythroid cells and ameliorating their survival. Furthermore, vamifeport improves the myeloid skewing in the MDS model, suggesting disease-modifying activity as single agent as well as combined therapy with luspatercept. Together, these data prove that combo therapies aimed at restricting iron and boosting erythroid maturation may offer additive beneficial effects in MDS and provide pre-clinical evidence for combining iron restriction and TFG-β superfamily ligand-trap approaches as more effective therapeutic strategies for the treatment of MDS.

Downregulation of Mitochondrial Complex II (MC II) in Myelodysplastic Syndromes

Myelodysplastic syndromes (MDS) are a group of diseases of hematopoietic stem cells (HSCs) that occur in the aging population and following therapy for unrelated cancers. The standard of care for MDS involves hypomethylating agents (HMAs) that invariably result in resistance and disease progression. We previously determined the role of mitochondrial metabolism in MDS pathophysiology, whereby suppression of mitochondrial activity leads to the accumulation of oncometabolites that competitively inhibit α-ketoglutarate-dependent dioxygenases (2OGDDs). Consequently, hypermethylation of epigenomes and hypoxia-inducible factor 1A (HIF1A) activation ensues a pseudohypoxia state that drives MDS pathogenesis. Our recent informatics analyses of primary MDS samples identified a patient subset with high TGF-β signaling in which mitochondrial complex II (MC II) genes were downregulated. MC II, or succinate dehydrogenase (SDH) is the smallest complex of the oxidative phosphorylation system that oxidizes succinate to fumarate and thereby donates electrons to reduce FAD to FADH 2 and the ubiquinone pool. Its downregulation results in the accumulation of Krebs cycle intermediate metabolites that can act as antagonists for 2OGDDs. To understand whether SDH dysregulation alone can contribute to MDS pathobiology, we generated a doxycycline-inducible and reversible mouse model (SDH-KD) that expresses shRNAs against the SDH subunits (SDHA, B, and D) in HSCs. These mice showed a gradual buildup of intracellular succinate in c-Kit + bone marrow (BM) cells and the peripheral blood, and a corresponding decrease of fumarate levels after induction for 2 weeks, 4 weeks, and 2 months. We found that knocking down Sdh was sufficient to induce pancytopenia and trilineage dysplasia in SDH-KD mice, while intracellular iron granules that surround the nucleus of erythroblasts, similar to ring sideroblasts commonly observed in MDS patients were also detected in BM and spleen smears. Of note, removing doxycycline and restoring Sdh expression rescued macrocytic anemia among other MDS phenotypes. Furthermore, treating SDH-KD mice with HMAs restored mitochondrial respiration and reversed HIF1A activation through the upregulation of SDH subunits. Using another MDS model, MllPTD/WT/ Vav1-Cre/Runx1 Flox/Flox, we found that HMA-treated HSCs differentiate towards CD11b +Gr1 + mature myeloid cells and that this requires Sdh expression. In conclusion, our results indicate that pseudohypoxia- and TGF-β signaling-associated changes in metabolic regulation caused by SDH downregulation underlie MDS pathogenesis. Elucidating the role of MC II in MDS pathobiology may provide alternative targets that can be combined with HMA or luspatercept therapy for MDS.

Myelodysplastic Syndrome Red Blood Cell Physiology Abnormalities and Their Correlation with Disease Risk and Therapeutic Interventions

PV and KT as well as VP and MA contributed equally Introduction: Myelodysplastic syndromes (MDS) are clonal hematopoietic disorders characterized by ineffective hematopoiesis, different degree of cytopenias, and increased risk of transformation to AML. Anemia constitutes a main clinical manifestation contributing to increased morbidity and mortality. It is currently managed with red blood cell (RBC) transfusions, erythropoiesis stimulating agents (EPO), and recently,with luspatercept. The latter enhances late stage erythropoiesis and is indicated for Ring Sideroblasts (RS) MDS resistant to EPO. Several lines of evidence suggest that MDS pathophysiology is associated with structural and functional distortions of mature red cell (RBCs), such as hemolysis that in rare instances include acquired lesions like hemoglobinopathies and membranopathies.These abnormalities add variability to the clinical MDS phenotypes and could serve as biomarkers for early diagnosis or monitoring. However, they have been scarcely investigated. Patients and methods: We examined 40 MDS patients and 12 healthy sex- and age-matched controls. According to the IPSS-Rrisk stratification system, 8 patients were High Risk (HR) and 32 patients Low Risk (LR)MDS, of whom 8 under EPO (EPO-LR) and 4 under luspatercept (luspatercept-LR) treatment. None of them had received RBC transfusions in the past three months. Following full blood count analyses and RBC fractionations, we measured hemolysis (basal, osmotic, mechanical, and oxidative), eryptosis and oxidative stress, as well as the antioxidant capacity of the plasma through spectrophotometry,fluorometry and flow cytometry. Results: Compared to controls, the cohort of MDS patients was characterized by anemia (Hb 10.7 vs 13.6g/dL) and significantly higher HDWand RDW (17.5 vs. 13.4%) indexes. RBCs exhibited signs of eryptosis (RBC calcium concentration 7063±1443 vs. 6027±1541 RFU/mg; PS surface exposure 0.61±0.45 vs. 0.33±0.17%) and oxidative stress (higher metHb/oxyHb ratio; membrane-bound hemichromes 45.0±15.3 vs. 30.2±13.3). About 18% of the patients presented spontaneous hemolysis. Clinical severity correlated with the levels of cytopenias, and oxidative hemolysis. Anemia correlated positively with the age, and the levels of membrane-bound hemichromes, eryptosis and intracellular reactive oxygen species (ROS). According to ROC analysis, the combination of RDW, metHb/oxyHb ratio, and total Hb concentration is predictive of MDS, across all IPSS-R categories(area under the curve: 0.908; Figure 1). We then examined the subgroups of MDS patients. Apart from the RDW, the LR-MDS subgroup exhibited on average normal RBC features, in opposite to the more anemic and thrombocytopenicHR-MDS subgroup that showed pathologically increasedresistance to osmotic hemolysis, intracellular calcium (7946±1917 vs. 6026±1541RFU), and metHb/oxyHb ratio (18.5±7.3 vs. 10.4±3.9%), when compared to controls. Of note, similar abnormalities were observed in the EPO subgroup that further showed reticulocytosis, PS externalization, and membrane-bound hemichromes(p<0.05) Compared to the non-treated subgroup, the EPO-LR had lower WBC counts, but higher RDW, PS exposure (0.75±0.44 vs. 0.41±0.26%) and resistance to osmotic stress, aspreviously reported in other EPO-treated patients.Luspatercept-LR had higher WBC and PLT counts but exhibited EPO-like effects on RBCs, including pathological resistance to osmotic/mechanical lysis, ROS concentration (1347±453 vs. 960±160 RFU), Hb oxidation, and PS exposure (1.05±0.60 vs. 0.41±0.26%), compared to the non-treated LR subgroup. Conclusion: MDS RBCs are highly heterogeneous in terms of volume and Hb concentration and present signs of oxidative stress and eryptosis. Consequently, these easily accessible markers could be an accessory tool to the MDS diagnosis.In the context of ineffective hematopoiesis and increased apoptosis of precursor erythroid cells,the LR-MDS patients exhibit minimal RBC abnormalities. On the other hand, the expansion of BM blasts and the lower level of apoptosis in HR-MDS seem to affect RBCs in a hemoglobinopathy-like manner in terms of Hb oxidation, eryptosis signaling, and membrane ion channels. EPO and luspatercept treated LR patients exhibit improved cytopenias but worse RBC physiology most probably attributed to clinical factors like RS phenotype.

Splicing factor mutations in the myelodysplastic syndromes: Role of key aberrantly spliced genes in disease pathophysiology and treatment.

Mutations of splicing factor genes (including SF3B1, SRSF2, U2AF1 and ZRSR2) occur in more than half of all patients with myelodysplastic syndromes (MDS), a heterogeneous group of myeloid neoplasms. Splicing factor mutations lead to aberrant pre-mRNA splicing of many genes, some of which have been shown in functional studies to impact on hematopoiesis and to contribute to the MDS phenotype. This clearly demonstrates that impaired spliceosome function plays an important role in MDS pathophysiology. Recent studies that harnessed the power of induced pluripotent stem cell (iPSC) and CRISPR/Cas9 gene editing technologies to generate new iPSC-based models of splicing factor mutant MDS, have further illuminated the role of key downstream target genes. The aberrantly spliced genes and the dysregulated pathways associated with splicing factor mutations in MDS represent potential new therapeutic targets. Emerging data has shown that IRAK4 is aberrantly spliced in SF3B1 and U2AF1 mutant MDS, leading to hyperactivation of NF-κB signaling. Pharmacological inhibition of IRAK4 has shown efficacy in pre-clinical studies and in MDS clinical trials, with higher response rates in patients with splicing factor mutations. Our increasing knowledge of the effects of splicing factor mutations in MDS is leading to the development of new treatments that may benefit patients harboring these mutations.

Prognostic Role of Cleaved Caspase-3 Reflecting Apoptosis in MDS Patients

Background: Myelodysplastic syndrome (MDS) is a heterogenous, clonal disorder in hematopoiesis. Pathophysiology of MDS is not clear yet, but there is growing interest in association with cellular proliferation and apoptosis. We hypothesized that Ki-67 and cleaved caspase-3 could be prognostic markers representative of proliferation and apoptosis in MDS patients. Methods: We performed retrospective study using bone marrow samples from 76 MDS patients who was diagnosed between 2004 and 2014 at Seoul National University Hospital. We compared Ki-67 and cleaved caspase-3 to evaluate cellular proliferative and apoptotic activities using immunohistochemical testing (IHC). And we additionally analyzed Ki-67 and cleaved caspase-3 between patients with MDS, idiopathic cytopenia of undetermined significance (ICUS) and health controls (HCs) based on data previously studied. And we also compared difference according to MDS risk scoring system. Results: Cleaved caspase-3 was highest in MDS patients, followed by ICUS patients and HCs (2.70, 95% CI: 2.56-2.85 vs. 2.19, 95% CI: 1.85-2.54 vs. 1.45, 95% CI: 1.17-1.73, p < 0.001). Similarly, the mean Ki-67 grade was also highest in MDS patients, followed by ICUS patients and HCs (1.72, 95% CI: 1.55-1.90 vs. 1.58, 95% CI: 1.30-1.86 vs 1.05, 95% CI: 0.95-1.16, p = 0.001) (Figure). Higher cleaved caspase-3 grade was significantly associated with lower IPSS-R score (p = 0.020), whereas Ki-67 was not. Interestingly, TET2 mutation was associated with decreased cleaved caspase-3 levels (p = 0.030). In terms of survival, there is no significant difference according to cleaved caspase-3 or Ki-67. In addition, we divided patients with MDS into four groups: lower-risk MDS patients with lower caspase-3 levels (A group : n = 32), lower-risk MDS patients with higher caspase-3 levels (B group : n = 13), higher-risk MDS patients with lower caspase-3 levels (C group : n = 28), and higher-risk MDS patients with higher caspase-3 levels (D group : n= 2). We compared PFS and OS between the four groups and found that high-risk MDS patients with lower caspase-3 showed significantly worse PFS than the other groups (p < 0.001; A vs B, p = 0.655; B vs C, p = 0.018; A vs C, p < 0.001) Conclusion: Our results suggest that apoptotic activity gradually increases from HCs, patients with ICUS to patients with MDS. Therefore, we could predict apoptotic activity from cleaved caspase-3. In addition, higher apoptotic activity was associated with better MDS patient prognostic scores and survival outcomes. Further studies are needed to reveal the the differences in apoptotic activity between lower- and higher-risk MDS. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Genetic Iron Overload Aggravates and Pharmacological Iron Restriction Improves MDS Pathophysiology in a Preclinical Mouse Model

Background: Patients with myelodysplastic syndromes (MDS) are prone to develop iron overload as a consequence of ineffective erythropoiesis and chronic transfusion therapy. Although iron overload is a common feature in MDS, it remains unclear whether and how iron excess is detrimental for MDS pathophysiology. Aims: This study aimed at characterizing altered iron homeostasis in a preclinical MDS mouse model, understanding the molecular mechanisms underlying iron toxicity and unraveling the potential therapeutic value of pharmacologic iron restriction in this disease condition. Methods: To this end, we took advantage of complementary approaches and analyzed the effect of iron overload obtained through genetic activation of the iron exporter ferroportin (FPNC326S), and iron restriction achieved through the administration of the FPN inhibitor vamifeport in NUP98-HOXD13 MDS mice, starting at 3 or 5 months of age. Results: At steady-state MDS mice develop an iron overload phenotype, hallmarked by low hepcidin levels, elevated serum iron and transferrin saturation, non-transferrin-bound iron (NTBI) formation and tissue iron deposition. This is associated with anemia and low WBCs in the peripheral blood. FPNC326S MDS mice show an aggravated iron phenotype, with further elevated NTBI and tissue iron accumulation and still inappropriately low hepcidin levels. Vamifeport administration in MDS mice reduced serum iron (194.8 vs 141.8 mg/dl; P<0.05) and NTBI formation (0.62 vs 0 μM; P<0.05) and prevented tissue iron loading. While iron excess in FPNC326S MDS mice did not improve erythropoiesis and hematologic parameters in the peripheral blood, iron restriction by vamifeport significantly ameliorated anemia (Hb 9.5 vs 11.4 g/dl; RBC 5.4 vs 6.9x106 cells/ml; P<0.05) and maturation of bone marrow as well as splenic RBCs in MDS mice. The improved ineffective erythropoiesis was associated with reduced oxidative stress and apoptosis in erythroid progenitors. Importantly, we observed a major role of the iron status in myeloid expansion in MDS. The number of immature myeloid blasts and myeloid progenitors in the bone marrow of MDS mice were aggravated by iron overload in FPNC326S MDS mice and attenuated by iron restriction in vamifeport-treated MDS mice compared to control MDS animals. Myeloid bias, monitored as percentage bone marrow myeloid cells, was exacerbated by iron overload (54.3 vs 43.1% bone marrow mononuclear cells - BMNCs) and alleviated by iron restriction (67.5 vs 74.8% BMNCs; P<0.05). This was associated with increased and reduced inflammatory activation of bone marrow macrophages in FPNC326S MDS and vamifeport-treated MDS mice, respectively. This observation demonstrates a role for NTBI in driving the reported pro-inflammatory activation of MDS monocytes/macrophages (Fuster JJ et al, Science 2017; Jaiswal S et al, NEJM 2017; Cull AH et al, Exp Hematol 2017) and suggests that this iron-driven mechanism contributes to myeloid expansion. Overall, these alterations translated into a faster and delayed transition to AML in iron-overloaded and iron-restricted MDS mice, respectively. Finally, iron overload aggravated and iron restriction alleviated ROS formation, DNA damage and pyroptotic cell death in HSPCs. By reducing the intracellular labile iron pool, vamifeport improved HSPC quality and survival, partially rescuing the stem cell pool size (0.16 vs 0.38% Lin-BMNCs; P<0.05) in MDS mice. The ameliorated HSPC pool, reduced myeloid bias and inflammation, and attenuated ineffective erythropoiesis resulted in improved hematologic parameters for more than 3 months, and a significant survival increment in iron-restricted compared to control MDS mice (>45 days; P=0.037). We further demonstrate that a subgroup of mice can benefit from late iron restriction by vamifeport administration at 5 months of age, resulting in improved anemia and prolonged survival. Conclusion: Our results show for the first time in a preclinical mouse model of MDS that iron excess driven by ineffective erythropoiesis has pathological implication in transfusion-independent MDS. These effects are likely aggravated by transfusions causing additional iron overload. Finally, iron restriction achieved through pharmacologic FPN inhibition by the oral FPN inhibitor vamifeport significantly improves MDS pathophysiology, uncovering the therapeutic potential of early prevention of NTBI formation in MDS.

Rker-050, a Novel Activin Receptor Type II Ligand Trap, Rescued Anemia and Reduced Bone Loss in a Mouse Model of Myelodysplastic Syndromes

Myelodysplastic syndromes (MDS) are a group of hematopoietic stem cell diseases characterized by ineffective hematopoiesis, peripheral blood cytopenias and dysplasias resulting from molecular defects and dysregulated signaling that govern the regulation of hematopoietic stem and progenitor cells (HSPCs). Crosstalk between cells in the osteo-hematopoietic (O-H) niche are necessary for the maintenance and self-renewal of HSPCs. Additionally, MDS patients commonly present with osteopenia and osteoporosis further demonstrating the critical interplay between the O-H cells in the bone marrow (BM). Alterations in the interaction between bone precursors and HSPCs within the BM microenvironment have been linked to the pathophysiology of MDS. Transforming growth factor-beta (TGF-β) superfamily signaling is instrumental in regulating both normal hematopoiesis and bone homeostasis. KER-050 is a modified investigational activin receptor type II ligand trap designed to bind and inhibit select TGF-β superfamily ligands, including activin A, activin B, GDF8 and GDF11, to promote hematopoiesis and bone growth. Preclinical studies have demonstrated that treatment with a research form of KER-050 (RKER-050) increased bone mass and multi-lineage hematopoiesis in healthy mice. To further investigate the effect of RKER-050 on hematopoietic and bone systems within the MDS disease setting, the NUP98/HOX13 MDS mouse model was utilized. Five-month-old male MDS mice were treated with vehicle (VEH, n=10), or RKER-050 (050, 7.5mg/kg, n=9) once weekly for 6 weeks along with a wildtype vehicle (WT, n=7) control group. To assess changes in hematological parameters, CBCs were measured, bone marrow (BM) and splenocytes were characterized via flow cytometry. To investigate alterations of bone microarchitecture, femurs were assessed using microCT imaging. MDS-VEH mice showed a reduction in red blood cells (RBC; 30%), hemoglobin (HGB; 19%), hematocrit (HCT; 13%) and reticulocytes (Retic; 16%) compared to WT; all hallmarks of human MDS. Relative to MDS-VEH, MDS-050 showed increased RBC, HGB, HCT and Retic by 25%, 13%, 10% and 39%, respectively. The erythroid Ter119 positive (Ter119+) cell population was slightly reduced in BM and spleen of MDS-VEH mice compared to WT. The BM Ter119+ cells were unchanged in the MDS-050 cohort relative to MDS-VEH; however, the spleen Ter119+ cells were increased similar to the levels in WT mice. Specific erythroid precursor populations were increased in BM and spleen of MDS-VEH mice versus WT, consistent with the ineffective erythropoiesis in this model. Interestingly, erythroid precursor populations were similar between MDS-VEH and MDS-050, yet MDS-050 exhibited recovered RBC production, suggesting an increase in maturation of erythroid precursors with RKER-050 treatment. Given the importance of the O-H niche in regulating hematopoiesis along with osteoporosis and osteopenia being comorbidities of MDS, bone microarchitecture was also analyzed. In the distal femoral trabecular bone, relative to WT mice, MDS-VEH mice had significant increases in trabecular (Tb) separation (33%) and decreases in Tb number (28%), changes that are consistent with reduced bone strength. Bone volume (34%) and bone volume fraction (26%) decreased but were not statistically significant. MDS-050 treated mice had a significant increase in bone volume (71%), higher bone volume fraction (88%), increased Tb number (44%) and decreased Tb spacing (20%) compared to the MDS-VEH, supporting RKER-050's role in improving bone mass. Taken together, these results suggest that RKER-050 can mitigate anemia and bone loss in an MDS mouse model, potentially by rebalancing hematopoiesis and bone turnover. In particular, the ability of RKER-050 to improve bone microarchitecture within the trabecular region, where the O-H niche resides, could be an important step in reestablishing healthy blood cell production. Furthermore, exploratory PD data from an ongoing phase 2 clinical trial demonstrate improved blood parameters with KER-050 treatment, including platelet and RBC in MDS patients (Tan et al. 2022 EHA #P776), and changes in bone biomarkers are currently being evaluated in the study. KER-050, thus, represents a potentially promising approach for patients with MDS and other hematological diseases, including myelofibrosis, where ineffective hematopoiesis and defects in bone homeostasis occur.

Upregulation of microRNA-597 in myelodysplastic syndromes induces apoptosis through FOSL2 inhibition.

Dysregulation of microRNAs (miRNAs) has been associated with the pathophysiology of myelodysplastic syndrome (MDS). Trisomy 8 is the most frequent chromosomal abnormalities in Korean patients with MDS. We investigated the dysregulation of miR-597-5p, located on chromosome 8, which is reported to induce cell death in numerous cancers. We compared the expression profiles of miR-597-5p among 65 MDS patients and 11 controls, and analyzed the in vitro effects of miR-597 on leukemic cells using an acute myeloid leukemia cell line transfected with miR-597. We found that miR-597-5p levels were upregulated 4.05-fold in MDS patients compared to those in controls. In vitro study results demonstrated that transfection with a miR-597 mimic induced apoptosis through downregulation of FOS like 2 (FOSL2). These findings suggest that upregulation of miR-597 induces apoptosis and that miR-597 has a possible role in the pathophysiology of MDS.

The Mesenchymal Niche in Myelodysplastic Syndromes.

Myelodysplastic syndromes (MDSs) are clonal disorders characterized by ineffective hematopoiesis, resulting in cytopenias and a risk of developing acute myeloid leukemia. In addition to mutations affecting hematopoietic stem cells (HSCs), numerous studies have highlighted the role of the bone marrow microenvironment (BMME) in the development of MDSs. The mesenchymal niche represents a key component of the BMME. In this review, we discuss the role of the mesenchymal niche in the pathophysiology of MDS and provide an overview of currently available in vitro and in vivo models that can be used to study the effects of the mesenchymal niche on HSCs.

Low-Dose Pesticides Alter Primary Human Bone Marrow Mesenchymal Stem/Stromal Cells through ALDH2 Inhibition.

Simple SummaryPesticide exposure is a public health concern. Two recent studies reported that exposure of normal primary bone marrow mesenchymal stem/stromal cells (BM-MSCs) to low-dose pesticide mixture induces deleterious consequences, such as oxidative stress, senescence, accelerated aging and increased sensitivity to oncogenic hits. Here, we show that exposure to this mixture decreases aldehyde dehydrogenase-2 (ALDH2) activity, with a concomitant increase in acetaldehyde level and DNA damage and alters the MSC capacity to support primitive hematopoiesis. Similar abnormalities were observed in bone marrow-MSCs from patients suffering from myelodysplastic syndrome (MDS), suggesting a role of pesticide-induced ALDH2 inhibition in the pathophysiology of this pre-leukemic disease.(1) Background: The impact of occupational exposure to high doses of pesticides on hematologic disorders is widely studied. Yet, lifelong exposure to low doses of pesticides, and more particularly their cocktail effect, although poorly known, could also participate to the development of such hematological diseases as myelodysplastic syndrome (MDS) in elderly patients. (2) Methods: In this study, a cocktail of seven pesticides frequently present in water and food (maneb, mancozeb, iprodione, imazalil, chlorpyrifos ethyl, diazinon and dimethoate), as determined by the European Food Safety Authority, were selected. Their in vitro effects at low-doses on primary BM-MSCs from healthy volunteers were examined. (3) Results: Exposure of normal BM-MSCs to pesticides for 21 days inhibited cell proliferation and promoted DNA damage and senescence. Concomitantly, these cells presented a decrease in aldehyde dehydrogenase 2 (ALDH2: mRNA, protein and enzymatic activity) and an increase in acetaldehyde levels. Pharmacological inhibition of ALDH2 with disulfiram recapitulated the alterations induced by exposure to low doses of pesticides. Moreover, BM-MSCs capacity to support primitive hematopoiesis was significantly altered. Similar biological abnormalities were found in primary BM-MSCs derived from MDS patients. (4) Conclusions: these results suggest that ALDH2 could participate in the pathophysiology of MDS in elderly people long exposed to low doses of pesticides.

Flow Cytometric Detection of the Ki-67 Proliferation Index and the Bcl-2 Anti-Apoptotic Index in Myelodysplastic Syndromes and Acute Myeloid Leukemia, and Their Clinical Implications

Introduction: Standardization of the detection and quantification of leukocyte differentiation markers by the EuroFlow Consortium has led to a major step forward in the integration of flow cytometry in classification of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). To further advance the integration and objectification of flow cytometry for characterization of these malignancies, more dynamic parameters assessing cell behavioral characteristics could prove useful, such as proliferative and (anti-)apoptotic markers. Proliferation and (anti-)apoptosis are processes that are tightly related to the pathogenesis, progression and chemo-/immunotherapy response of cancers. As a result, proliferation and (anti-)apoptotic markers have proven their value as objective parameters in the field of histopathology for diagnostic and prognostic applications in solid tumors and lymphoma. Although use of proliferative and (anti-)apoptotic markers as objective parameters in the diagnostic process of MDS and AML was studied in the past decades, this did not result in the incorporation of these biomarkers in their clinical diagnosis. The recent developments in flow cytometric analyses now allow the quantification of proliferative and (anti-)apoptotic fractions at the level of individual maturing bone marrow cells. Therefore, we aim to determine the Ki-67 proliferation indices and Bcl-2 anti-apoptotic indices in maturing bone marrow cells in order to assess whether these parameters could have future clinical implications for the diagnostic work-up of MDS and AML.Methods: Fifty bone marrow aspirates from femoral heads of non-malignant cases, 20 aspirates of MDS patients and 20 aspirates of AML were included in this study. Ten-color flow cytometry in combination with a software-based maturation tool was used for analysis of the Ki-67 proliferative and Bcl-2 anti-apoptotic indices of blast cells and during the erythro-, myelo-, and monopoiesis.Results: Ki-67 proliferative indices of blast cells and immature erythroid, myeloid and monocytic cells were significantly lower in MDS patients compared to the non-malignant cases, while the Bcl-2 anti-apoptotic indices were significantly elevated in these cells. Furthermore, the Bcl-2 anti-apoptotic indices were also increased in mature erythroid, myeloid and monocytic cells of MDS patients. The decreased Ki-67 proliferative indices and increased Bcl-2 anti-apoptotic indices in blast cells and erythroid, myeloid and monocytic cells were even more prominently observed in AML patients.Conclusions: The lowered Ki-67 proliferative indices and elevated Bcl-2 anti-apoptotic indices in blast cells and immature progenitor cells led to a better understanding of the pathophysiology of MDS and AML, and explained the low chemotherapy response of these patients. Side-effects of such therapies can also be explained by the Ki-67 proliferation indices and Bcl-2 anti-apoptotic indices. Moreover, the increase of the Bcl-2 anti-apoptotic fraction is an important factor in the progression of MDS to AML. Future studies on the clinical applications of these parameters for MDS and AML are necessary and can include many applications, such as prediction of chemo-/immunotherapy response, diagnostic and prognostic applications. DisclosuresRamaekers: Nordic-MUbio: Current Employment.

From Immune Dysregulations to Therapeutic Perspectives in Myelodysplastic Syndromes: A Review.

The pathophysiology of myelodysplastic syndromes (MDSs) is complex and often includes immune dysregulation of both the innate and adaptive immune systems. Whereas clonal selection mainly involves smoldering inflammation, a cellular immunity dysfunction leads to increased apoptosis and blast proliferation. Addressing immune dysregulations in MDS is a recent concept that has allowed the identification of new therapeutic targets. Several approaches targeting the different actors of the immune system have therefore been developed. However, the results are very heterogeneous, indicating the need to improve our understanding of the disease and interactions between chronic inflammation, adaptive dysfunction, and somatic mutations. This review highlights current knowledge of the role of immune dysregulation in MDS pathophysiology and the field of new drugs.

Pathophysiology of Myelodysplastic Syndromes

Ineffective hematopoiesis is the major characteristic of early myelodysplastic syndromes. Its pathophysiology relies on a diversity of mechanisms supported by genetic events that develop in aging hematopoietic stem cells. Deletion and mutations trigger epigenetic modifications, and co-transcriptional and post-transcriptional deregulations of gene expression. Epistatic interactions between mutants may aggravate the phenotype. Amplification of minor subclones containing mutations that promote their growth and suppress the others drives the clonal evolution. Aging also participates in reprogramming the immune microenvironment towards an inflammatory state, which precedes the expansion of immunosuppressive cells such as Tregs and myeloid-derived suppressive cells that alters the anti-tumor response of effector cells. Integrating biomarkers of transcription/translation deregulation and immune contexture will help the design of personalized treatments.

Myelodysplastic Syndromes in the Postgenomic Era and Future Perspectives for Precision Medicine.

Simple SummaryWith demographic ageing, improved cancer survivorship and increased diagnostic sensitivity, incident cases of patients with Myelodysplastic Syndromes (MDS) are continuously rising, leading to a relevant impact on health care resources. Disease heterogeneity and various comorbidities are challenges for the management of the generally elderly patients. Therefore, experienced physicians and multidisciplinary teams should be involved in the establishment of the correct diagnosis, risk-assessment and personalized treatment plan. Next-generation sequencing allows for early detection of clonal hematopoiesis and monitoring of clonal evolution, but also poses new challenges for its appropriate use. At present, allogeneic hematopoietic stem cell transplantation remains the only curative treatment option for a minority of fit MDS patients. All others receive palliative treatment and will eventually progress, having an unmet need for novel therapies. Targeting compounds are in prospect for precision medicine, however, abrogation of clonal evolution to acute myeloid leukemia remains actually out of reach.Myelodysplastic syndromes (MDS) represent a heterogeneous group of clonal disorders caused by sequential accumulation of somatic driver mutations in hematopoietic stem and progenitor cells (HSPCs). MDS is characterized by ineffective hematopoiesis with cytopenia, dysplasia, inflammation, and a variable risk of transformation into secondary acute myeloid leukemia. The advent of next-generation sequencing has revolutionized our understanding of the genetic basis of the disease. Nevertheless, the biology of clonal evolution remains poorly understood, and the stochastic genetic drift with sequential accumulation of genetic hits in HSPCs is individual, highly dynamic and hardly predictable. These continuously moving genetic targets pose substantial challenges for the implementation of precision medicine, which aims to maximize efficacy with minimal toxicity of treatments. In the current postgenomic era, allogeneic hematopoietic stem cell transplantation remains the only curative option for younger and fit MDS patients. For all unfit patients, regeneration of HSPCs stays out of reach and all available therapies remain palliative, which will eventually lead to refractoriness and progression. In this review, we summarize the recent advances in our understanding of MDS pathophysiology and its impact on diagnosis, risk-assessment and disease monitoring. Moreover, we present ongoing clinical trials with targeting compounds and highlight future perspectives for precision medicine.

Oxidative Stress in Stem / Progenitor Cells and Specific Antioxidant Response in Bone Marrow from Myelodysplastic Syndrome Patients

Introduction: Myelodysplastic syndromes (MDS) are heterogeneous group of blood diseases with varying degrees of severity, which can be classified into one of several subgroups based on features of the abnormal cells. The progression of MDS to secondary AML (sAML) is a good example of the multi-step theory of leukemogenesis in which a series of mutations occur in an initially normal cell and transform it into a cancer cell. Reactive Oxygen Species (ROS) are highly involved in normal hematopoiesis, a low amount being associated with quiescence and self-renewal, while a higher level is requested for ROS signaling pathways. The level of intracellular ROS is finely tuned by the expression of ROS-related enzymes that are suggested as major players in human MDS / AML. Altogether, these elements highlight the potential implication of redox metabolism in the pathophysiology of MDS / sAML.Methods: This study focused on ROS level and antioxidant gene expression in bone marrow (BM) cells from 97 patients compared to 25 healthy controls. All subjects were informed and consenting following a procedure approved by the ethical committee and provided a written informed consent. sAML patients were characterized at diagnosis by proliferation of blasts (>20%) and dysplasia in BM samples. The sternal BM samples were obtained at the time of diagnosis (University hospital of Tours and Cochin hospital in Paris), and were compared with BM from aged-matched healthy donors. Flow cytometry analyses and RNA extraction were processed within the first hour following BM aspiration. We quantified: (i) the level of ROS by flow cytometry (DCFDA staining) in the subpopulations of BM cells identified with anti-CD45-APC-H7, anti-CD34-PE-Cy7 and anti-CD38-APC antibodies; (ii) the expression level of 28 transcripts encoding for major enzymes implicated in the antioxidant cellular response by qRT-PCR using Universal Probe Library technology. All statistical analyses were conducted using R v3.2.2 software. Differences in ROS levels between pathologies was tested using Kruskall-Wallis test followed by Dunn's post hoc test. Differences in expression data between patients and controls were tested using Mann-Whitney test on ΔCt and RQ. Principal component analyses were performed using FactoMineR package on ΔCt values. Significant differences were identified on a change of at least 2-fold and P value less than .05.Results: The results highlight increased ROS level in BM non-lymphoid cells, especially in CD34high CD38low stem and progenitor cells. This increase is also much important in case of sAML compared to low grade (MDS-SLD and MDS-MLD) or high grade MDS (MDS-EB1 and MDS-EB2). Interestingly and as expected, the most important oxidative stress was found in erythroblast subpopulation in MDS-SLD-RS and MDS-MLD-RS, enriched in ring sideroblasts. Moreover, we identified a specific antioxidant signature in the different MDS / sAML bone marrow samples. More precisely, the progression of the disease was characterized by 3-step antioxidant cellular response: (i) overexpression of enzymes reducing disulfides bonds in protein (low-grade MDS - GLRX family), then (ii) enzymes detoxifying H2O2 (high-grade MDS - PRDX and GPX families) and finally (iii) decreased expression of these enzymes in sAML.Conclusion: We report for the first-time progressive increase in ROS level in CD34posCD38low stem andprogenitor cells from MDS patients compared to healthy controls, and higher level in sAML compared to MDS patients. In the light of these findings, antioxidant treatments should be explored in MDS with low blast counts, to avoid the risk of promoting transformation. Concomitantly, MDS BM cells initiate antioxidant program by overexpressing enzymes reducing disulfide bonds in proteins (GLRX family) then reinforced by H2O2 detoxifying enzymes (PRDX and GPX families), these enzymes being dramatically decreased when transformation in sAML. This specific molecular antioxidant response could be considered as biomarkers useful for diagnosis and follow-up of the disease. DisclosuresFontenay:Celgene: Research Funding.

TET2 Deficiency Led to Expansion of Dysfunctional Erythroid Progenitors

Erythrocyte development is a complex process involving multiple developmental states that include erythroid progenitors, burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) cells and erythroid precursors, proerythroblasts, basophilic erythroblasts, polychromatic erythroblasts and orthochromatic erythroblasts in the bone marrow. Orthochromatic erythroblasts expel nucleus to become reticulocytes which mature into erythrocytes in blood stream. Defects at any of these developmental stages will lead to anemia. Myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell disorders characterized by cytopenia and ineffective hematopoiesis. Anemia is the defining cytopenia in the majority of patients with MDS, yet the molecular mechanisms for dyserythropoiesis in MDS remain largely unclear. Recent studies have revealed that heterozygous loss-of-function mutation of DNA dioxygenase TET2 is the most common mutation in MDS.In the present study, we explored shRNA-mediated knockdown approach in human CD34+ cell to study the role of TET2 in human erythropoiesis. We show that TET2 is downregulated starting at the CFU-E stage. Growth curves of purified CFU-E cells show no difference between control and TET2 knockdown cells until day 6. An increased proliferation of TET2 knockdown-CFU-E cells is noted starting at day 7. Interestingly, while the growth of control CFU-E cells plateaued on day 11 with completion of terminal erythroid differentiation and enucleation, TET2 knockdown CFU-E cells continued their growth for additional 13 days. Flow cytometry and cytospin morphologic analyses revealed that by day 11 all control cells differentiated into GPA+ late stage erythroblasts or reticulocytes. In marked contrast, about 30 % of TET2 knockdown cells are still GPA- but CD71+, IL-3R-, CD34-, CD36+, phenotypically resembling erythroid progenitor CFU-E cells. Surprisingly, in contrast to freshly purified control and TET2 knockdown CFU-E cells that generate colonies in methylcellulose colony forming assays, these cells fail to give rise to CFU-E colonies. We thus termed these late proliferating cells as “TET2 knockdown marker CFU-E”. Further characterization of the “TET2 knockdown marker CFU-E” cells revealed that in contrast to control CFU-E, the “TET2 knockdown marker CFU-E” exhibit following distinct features: 1) they had lower proliferative potential; 2) their proliferation required both EPO and SCF (while control CFU-E cells only require EPO); 3) they underwent extensive apoptosis in the absence of SCF; 4) they failed to undergo terminal erythroid differentiation. These findings demonstrate that TET2 knockdown led to expansion of dysfunctional erythroid progenitors.To explore the underlying molecular mechanisms, we performed RNA-seq analysis on control CFU-E, TET2 knockdown CFU-E and “TET2 knockdown marker CFU-E” cells. Bioinformatics analysis revealed that while only about 50 genes were differentially expressed between control CFU-E and TET2 knockdown CFU-E, about 400 genes were differentially expressed between control CFU-E and “TET2 knockdown marker CFU-E”. Gene set enrichment analysis revealed upregulation of pathways associated with apoptosis/cell death. Notably, FAS (CD95), the cell surface death receptor previously shown to be upregulated in MDS bone marrow cells, was among the top five upregulated apoptosis associated genes. The increased surface expression of FAS was also noted in TET2-knockdown cells as assessed by flow cytometry. Furthermore, the increased surface expression of FAS was detected in primary bone marrow erythroblasts of MDS patients with TET2 mutation. Our findings uncovered previously unknown roles of TET2 and its potential underlying molecular mechanisms. As the phenotypic changes upon TET2 knockdown resemble the characteristic pathological changes of MDS, our findings suggest the involvement of TET2 deficiency in the pathophysiology of MDS. DisclosuresAli:Onconova Therapeutics: Consultancy; Kura Oncology: Consultancy. Raza:Celgene Inc.: Research Funding; Syros Pharmaceuticals: Research Funding; Onconova Therapeutics: Research Funding, Speakers Bureau; Genoptix: Speakers Bureau; Kura Oncology: Research Funding; Novartis: Speakers Bureau; Janssen R&D: Research Funding.