Gisela Caceres

Lenalidomide Promotes p53 Degradation by Inhibiting MDM2 Auto-ubiquitination in Myelodysplastic Syndrome with Chromosome 5q Deletion

Allelic deletion of the RPS14 gene is a key effector of the hypoplastic anemia in patients with myelodysplastic syndrome (MDS) and chromosome 5q deletion (del(5q)). Disruption of ribosome integrity liberates free ribosomal proteins to bind to and trigger degradation of mouse double minute 2 protein (MDM2), with consequent p53 transactivation. Herein we show that p53 is overexpressed in erythroid precursors of primary bone marrow del(5q) MDS specimens accompanied by reduced cellular MDM2. More importantly, we show that lenalidomide (Len) acts to stabilize MDM2, thereby accelerating p53 degradation. Biochemical and molecular analyses showed that Len inhibits the haplodeficient protein phosphatase 2A catalytic domain alpha (PP2Acα) phosphatase resulting in hyperphosphorylation of inhibitory serine-166 and serine-186 residues on MDM2, and displaces binding of RPS14 to suppress MDM2 autoubiquitination whereas PP2Acα overexpression promotes drug resistance. Bone marrow specimens from del(5q) MDS patients resistant to Len overexpressed PP2Acα accompanied by restored accumulation of p53 in erythroid precursors. Our findings indicate that Len restores MDM2 functionality in the 5q- syndrome to overcome p53 activation in response to nucleolar stress, and therefore may warrant investigation in other disorders of ribosomal biogenesis.

Identification of a risk dependent microRNA expression signature in myelodysplastic syndromes

The myelodysplastic syndromes (MDS) display both haematological and biological heterogeneity with variable leukaemia potential. MicroRNAs play an important role in tumour suppression and the regulation of self‐renewal and differentiation of haematopoietic progenitors. Using a microarray platform, we evaluated microRNA expression from 44 patients with MDS and 17 normal controls. We identified a thirteen microRNA signature with statistically significant differential expression between normal and MDS specimens (P < 0·01), including down‐regulation of members of the leukaemia‐associated MIRLET7 family. A unique signature consisting of 10 microRNAs was closely associated with International Prognostic Scoring System (IPSS) risk category permitting discrimination between lower (Low/Intermediate‐1) and higher risk (Intermediate‐2/High) disease (P < 0·01). Selective overexpression of MIR181 family members was detected in higher risk MDS, indicating pathogenetic overlap with acute myeloid leukaemia. Survival analysis of an independent cohort of 22 IPSS lower risk MDS patients revealed a median survival of 3·5 years in patients with high expression of MIR181 family compared to 9·3 years in patients with low MIR181 expression (P = 0·002). Our pilot study suggested that analysis of microRNA expression profile offers diagnostic utility, and provide pathogenetic and prognostic discrimination in MDS.

TP53 suppression promotes erythropoiesis in del(5q) MDS, suggesting a targeted therapeutic strategy in lenalidomide-resistant patients.

Significance The hypoplastic anemia characteristic of del(5q) myelodysplastic syndrome (MDS) arises from ribosomal protein insufficiency, resulting in erythroid-specific activation of p53. We found that suppression of p53 by cenersen, an antisense oligonucleotide, markedly improved erythroid colony formation in primary MDS specimens assessed by two-stage colony formation assay. Erythropoietic rescue significantly correlated with the magnitude of reduction in nuclear p53. In addition, in a cohort of eight lower-risk, lenalidomide-refractory del(5q) MDS patients treated with lenalidomide and dexamethasone, a glucocorticoid receptor-dependent antagonist of p53, transfusion independence was restored in five of eight patients, accompanied by in vivo expansion of erythroid precursors without clonal suppression. These results suggest inhibition of p53 may be a unique therapeutic strategy in patients with lenalidomide-resistant del(5q) MDS. Stabilization of p53 in erythroid precursors in response to nucleosomal stress underlies the hypoplastic anemia in myelodysplastic syndromes (MDS) with chromosome 5q deletion [del(5q)]. We investigated whether cenersen, a clinically active 20-mer antisense oligonucleotide complementary to TP53 exon10, could suppress p53 expression and restore erythropoiesis in del(5q) MDS. Cenersen treatment of ribosomal protein S-14-deficient erythroblasts significantly reduced cellular p53 and p53-up-regulated modulator of apoptosis expression compared with controls, accompanied by a significant reduction in apoptosis and increased cell proliferation. In a two-stage erythroid differentiation assay, cenersen significantly suppressed nuclear p53 in bone marrow CD34+ cells isolated from patients with del(5q) MDS, whereas erythroid burst recovery increased proportionally to the magnitude of p53 suppression without evidence of del(5q) clonal suppression (r = −0.6; P = 0.005). To explore the effect of p53 suppression on erythropoiesis in vivo, dexamethasone, a glucocorticoid receptor-dependent p53 antagonist, was added to lenalidomide treatment in eight lower-risk, transfusion-dependent, del(5q) MDS patients with acquired drug resistance. Transfusion independence was restored in five patients accompanied by expansion of erythroid precursors and decreased cellular p53 expression. We conclude that targeted suppression of p53 could support effective erythropoiesis in lenalidomide-resistant del(5q) MDS.

Paroxysmal nocturnal hemoglobinuria and concurrent JAK2(V617F) mutation.

Paroxysmal nocturnal hemoglobinuria (PNH) is a hemolytic and pro-thrombotic disorder associated with the clonal expansion of hematopoietic stem cells harboring somatic mutations in the PIG-A gene.1 Mutations in PIG-A result in a lack of surface expression of all glycosylphosphatidylinositol (GPI)-anchored proteins, including the complement inhibitors CD55 and CD59,2 which is responsible for the hemolytic (and probably also the pro-thrombotic) phenotype.3, 4 However, long-term colony-forming assays and in vivo murine models have failed to show that PIG-A mutations are alone sufficient to drive clonal expansion.5, 6 There are two leading hypotheses to account for clonal expansion of PIG-A-null stem cells: (i) clonal selection and (ii) second mutations. The first model posits that aplastic anemia—which is epidemiologically associated with PNH—results in an immune-mediated marrow injury that selectively spares PNH stem cells.7 The second hypothesis is borrowed from models of oncogenesis and may be supported by the observation that patients with PNH can harbor clonal cytogenetic abnormalities.8 However, no specific gene mutations other than PIG-A have been reported in patients with PNH—with one exception. In two patients, a rearrangement of chromosome 12 with a break in the 3′-untranslated region of the HMGA2 gene has been reported.9 Overexpression of an HMGA2 truncated protein recapitulates an myeloproliferative neoplasms (MPN)-like phenotype in a murine model and could theoretically contribute to clonal expansion in PNH.10 Interestingly, literature from the 1970s has reported several cases of PNH in association with myelofibrosis and other MPNs.11, 12 Here we report on three index cases of PNH with myeloproliferative features harboring a JAK2V617F mutation, which is now understood to drive clonal expansion in many MPNs.13 The first case is a 51-year-old male, presenting with right hemiparesis and dysarthria secondary to a stroke, followed by multiple thrombotic events, including the Budd Chiari Syndrome (BCS). A hypercoagulable workup revealed the presence of the JAK2V617F mutation in the peripheral blood. Upon referral to us, a complete blood count noted an Hgb of 5 g/dl, platelets (PLTs) of 492 × 109/l and a white blood cell (WBC) 8.90 × 109/l. Bone marrow biopsy revealed a hypercellular marrow (80–100%), dysmegakaryopoiesis, a 4% myeloblast population and normal cytogenetics (Figure 1). The patient later presented with an elevated lactate dehydrogenase (LDH) and undetectable haptoglobin. Flow cytometry revealed that 99% of the granulocytes and 13% of the erythrocytes were GPI (−), confirming the diagnosis of PNH, and the patient was initiated on eculizumab. Although he has been transfusion independent for 3 years with this therapy, he has had multiple complications of BCS, including esophageal variceal hemorrhage. Figure 1 Morphological features of the bone marrow in patients with PNH and the JAK2V617F mutation. Patient 1: (i) hematoxilin–eosin-stained section showing hyperplasia and mild dysplasia. (ii) Anti-factor VIII section showing increased number of megakaryocytes ... The second patient, a 65-year-old male, presented with darkened urine and transfusion-dependent anemia, and was found to have 40% PNH red cells. At that time, hematological parameters were: WBC 6.7 × 109/l, 76.7% granulocytes Hgb 95 g/l and PLTs 580 × 109/l. His LDH was nine-fold the upper limit of normal. Two years later, despite prophylactic anticoagulation with coumadin, he developed a splenic infarction, prompting a splenectomy. His WBC then rose to over 100 000 × 109/l and his PLT count to over 1 000 000 × 109/l, and the JAK2V617F mutation was identified. He developed a post-operative portal vein thrombosis, and he was treated at that time with hydroxyurea, oral anticoagulation and eculizumab. Ten months later, he developed BCS in the setting of a therapeutic international normalized ratio, but low trough eculizumab levels; BCS completely resolved with intravenous tissue plasminogen activator, and he was started on aspirin, fondaparinux and hydroxyurea—and a shorter eculizumab-dosing interval. He then developed a progressively increasing WBC, and the marrow demonstrated hypercellularity, granulocytic hyperplasia and moderate reticulin fibrosis (Figure 1), and a 46, XY, del (17) (p11) (18/24) karyotype. Three additional metaphases demonstrated the del17 abnormality with a non-clonal additional abnormality in each case. He was given decitabine, but expired 4 years after the diagnosis of PNH secondary to progressive liver failure due to iron overload. The third patient is a 78-year-old male with a history of prostate cancer in 2004, treated with brachytherapy. He also had a history of arthritis, pulmonary hypertension and cutaneous melanoma. In 2003, he was mildly anemic, and in 2006, a marrow examination revealed hypercellularity without excess blasts. In 2009, he noticed dark urine; the Hgb was 102 g/l with 4.5% reticulocytes, PLTs 648 × 109/l, WBC 10.1 × 109/l, 77% polys, 15% lymphocytes and 7% monocytes. The LDH was 1367 IU/L (normal <243), and flow cytometry demonstrated 73% CD59-negative granulocytes and 53% CD59-negative red cells. Repeat marrow examination in 2010 was notable for hypercellularity and severe fibrosis (Figure 1). The karyotype was normal (20/20), and the JAK2V617F mutation was identified. The patient developed transfusion-dependent anemia not responding to eculizumab, steroids or danazol. At the time of referral in 2011, the WBC was 12.1 × 109/l with 71% polys, 3% metamyelocytes, 2% myelocytes, 1% blasts, 10% lymphocytes, 8% monocytes, 3% basophils and 1% nucleated red blood cells; Hgb was 79 g/l and the absolute reticulocyte count was 97 500/μl. The peripheral blood smear demonstrated poikilocytosis, rare teardrops, some giant PLTs and giant early myeloid precursors. LDH was 1047 IU/l (normal <180) and ferritin was 2908 ng/ml. The spleen was not palpable. Repeat analysis demonstrated 43% CD59-negative red cells and 99.7% FLAER-negative, CD24-negative granulocytes. Molecular testing again revealed the JAK2V617F mutation, and he was treated with eculizumab, aspirin and fondaparinux. The patient had several bouts of pneumonia and eventually succumbed in September 2011 to a severe Clostridium difficile infection. A total of 26 subsequent patients (mean age, 36 years; range, 15–65) with PNH were tested for the JAK2V617F mutation by the DNA tetra-primer amplification refractory mutation system (ARMS). All patients had intravascular hemolysis and were considered to have classic PNH. A total of 23 cases (77%) were de novo and 7 (23%) had antecedent aplastic anemia. The percentage of GPI (−) granulocytes ranged from 22 to 99% (median 71.6%) and GPI (−) erythrocytes ranged from 0.86 to 72.3% (median 32.4%) by FLAER staining and flow cytometry. Serum LDH levels were elevated in most patients (mean 1204 IU/l, range 129–4393 IU/l, normal <618 IU/l). The median WBC was 4.48 × 109/l (range, 2.44–8.90), median Hgb level was 117 g/l (range, 6.4–14.0) and median PLT count was 108 × 109/l (range, 28–648). Hepatic, cerebral or mesenteric thrombosis was found in 6 of the 29 patients (21%), and 3 had BCS. On the basis of the ARMS analysis, the JAK2V617F mutation was not detected in any of these 26 patients. In both case 1 and 2, the molecular lesion in the PIG-A gene was identified to be a 500-kb deletion at Xp22.2 by single-nucleotide polymorphism array, as we have published previously.14 To determine the cellular origin of the JAK2V617F mutation in these two patients, DNA was extracted from GPI (−) CD11b+ granulocytes, GPI (+) CD11b+ granulocytes and CD3+ T cells after FLAER staining and flow cytometry sorting. JAK2V617F was selectively detected in GPI (−) granulocytes, but not in GPI (+) granulocytes or T cells, clearly showing that the JAK2V617F mutation is not in the germline and that it co-exists within in the PNH clone (Figure 2). Figure 2 Cellular origin of PNH and JAK2V617F mutation. (a) To clarify the cellular origin of JAK2V617F mutation, GPI (−) cells and GPI (+) cells were isolated with fluorescence-activated cell-sorting method using a FACSAria (BD Biosciences, San ... The occurrence of PNH clones in MDS and aplastic anemia is well documented and routinely evaluated in clinical practice. This series now documents the coexistence of PNH and JAK2V617F-associated MPNs in three index cases with a higher PLT count and WBC compared with PNH patients overall (Figure 2b). Two of the three cases were shown to harbor the JAK2V617F mutation within the PNH clone. In the third patient, 99% of the granulocytes were GPI (−), suggesting that JAK2V617F mutation also occurred within the PNH clone. Apart from HMGA2, this now represents the second mutation coexisting with PNH that could explain clonal expansion. The prevalence of PNH clones among MPN patients is unknown, because these three index patients were ascertained by referral. In rare patients, even before the era of flow cytometry and molecular testing, a MPN/PNH overlap syndrome was identified.11, 13, 14 Of note, no JAK2V617F mutations were detected in 26 subsequently screened PNH patients, suggesting that the prevalence of this mutation is not high in classic PNH, consistent with an earlier report.15 On the basis of these findings, we believe that JAK2 mutation testing should be performed on patients with PNH, who have elevated peripheral blood counts. Considering that most patients with PNH are mildly cytopenic (Figure 2b), this recommendation might extend to those with blood counts towards the upper limit of normal. Unexplained splenomegaly or a fibrotic marrow would also represent an indication for JAK2 mutation analysis. Conversely, patients with an MPN and signs of hemolysis should be tested for PNH by flow cytometry.

Erythropoietin receptor signaling is membrane raft dependent.

Upon erythropoietin (Epo) engagement, Epo-receptor (R) homodimerizes to activate JAK2 and Lyn, which phosphorylate STAT5. Although recent investigations have identified key negative regulators of Epo-R signaling, little is known about the role of membrane localization in controlling receptor signal fidelity. Here we show a critical role for membrane raft (MR) microdomains in creation of discrete signaling platforms essential for Epo-R signaling. Treatment of UT7 cells with Epo induced MR assembly and coalescence. Confocal microscopy showed that raft aggregates significantly increased after Epo stimulation (mean, 4.3±1.4(SE) vs. 25.6±3.2 aggregates/cell; p≤0.001), accompanied by a >3-fold increase in cluster size (p≤0.001). Raft fraction immunoblotting showed Epo-R translocation to MR after Epo stimulation and was confirmed by fluorescence microscopy in Epo stimulated UT7 cells and primary erythroid bursts. Receptor recruitment into MR was accompanied by incorporation of JAK2, Lyn, and STAT5 and their activated forms. Raft disruption by cholesterol depletion extinguished Epo induced Jak2, STAT5, Akt and MAPK phosphorylation in UT7 cells and erythroid progenitors. Furthermore, inhibition of the Rho GTPases Rac1 or RhoA blocked receptor recruitment into raft fractions, indicating a role for these GTPases in receptor trafficking. These data establish a critical role for MR in recruitment and assembly of Epo-R and signal intermediates into discrete membrane signaling units.

Lenalidomide Stabilizes the Erythropoietin Receptor by Inhibiting the E3 Ubiquitin Ligase RNF41

In a subset of patients with non-del(5q) myelodysplastic syndrome (MDS), lenalidomide promotes erythroid lineage competence and effective erythropoiesis. To determine the mechanism by which lenalidomide promotes erythropoiesis, we investigated its action on erythropoietin receptor (EpoR) cellular dynamics. Lenalidomide upregulated expression and stability of JAK2-associated EpoR in UT7 erythroid cells and primary CD71+ erythroid progenitors. The effects of lenalidomide on receptor turnover were Type I cytokine receptor specific, as evidenced by coregulation of the IL3-Rα receptor but not c-Kit. To elucidate this mechanism, we investigated the effects of lenalidomide on the E3 ubiquitin ligase RNF41. Lenalidomide promoted EpoR/RNF41 association and inhibited RNF41 auto-ubiquitination, accompanied by a reduction in EpoR ubiquitination. To confirm that RNF41 is the principal target responsible for EpoR stabilization, HEK293T cells were transfected with EpoR and/or RNF41 gene expression vectors. Steady-state EpoR expression was reduced in EpoR/RNF41 cells, whereas EpoR upregulation by lenalidomide was abrogated, indicating that cellular RNF41 is a critical determinant of drug-induced receptor modulation. Notably, shRNA suppression of CRBN gene expression failed to alter EpoR upregulation, indicating that drug-induced receptor modulation is independent of cereblon. Immunohistochemical staining showed that RNF41 expression decreased in primary erythroid cells of lenalidomide-responding patients, suggesting that cellular RNF41 expression merits investigation as a biomarker for lenalidomide response. Our findings indicate that lenalidomide has E3 ubiquitin ligase inhibitory effects that extend to RNF41 and that inhibition of RNF41 auto-ubiquitination promotes membrane accumulation of signaling competent JAK2/EpoR complexes that augment Epo responsiveness. Cancer Res; 76(12); 3531-40. ©2016 AACR.

Lenalidomide induces lipid raft assembly to enhance erythropoietin receptor signaling in myelodysplastic syndrome progenitors.

Anemia remains the principal management challenge for patients with lower risk Myelodysplastic Syndromes (MDS). Despite appropriate cytokine production and cellular receptor display, erythropoietin receptor (EpoR) signaling is impaired. We reported that EpoR signaling is dependent upon receptor localization within lipid raft microdomains, and that disruption of raft integrity abolishes signaling capacity. Here, we show that MDS erythroid progenitors display markedly diminished raft assembly and smaller raft aggregates compared to normal controls (p = 0.005, raft number; p = 0.023, raft size). Because lenalidomide triggers raft coalescence in T-lymphocytes promoting immune synapse formation, we assessed effects of lenalidomide on raft assembly in MDS erythroid precursors and UT7 cells. Lenalidomide treatment rapidly induced lipid raft formation accompanied by EpoR recruitment into raft fractions together with STAT5, JAK2, and Lyn kinase. The JAK2 phosphatase, CD45, a key negative regulator of EpoR signaling, was displaced from raft fractions. Lenalidomide treatment prior to Epo stimulation enhanced both JAK2 and STAT5 phosphorylation in UT7 and primary MDS erythroid progenitors, accompanied by increased STAT5 DNA binding in UT7 cells, and increased erythroid colony forming capacity in both UT7 and primary cells. Raft induction was associated with F-actin polymerization, which was blocked by Rho kinase inhibition. These data indicate that deficient raft integrity impairs EpoR signaling, and provides a novel strategy to enhance EpoR signal fidelity in non-del(5q) MDS.

HG-829 is a potent noncompetitive inhibitor of the ATP-binding cassette multidrug resistance transporter ABCB1.

Transmembrane drug export mediated by the ATP-binding cassette (ABC) transporter P-glycoprotein contributes to clinical resistance to antineoplastics. In this study, we identified the substituted quinoline HG-829 as a novel, noncompetitive, and potent P-glycoprotein inhibitor that overcomes in vitro and in vivo drug resistance. We found that nontoxic concentrations of HG-829 restored sensitivity to P-glycoprotein oncolytic substrates. In ABCB1-overexpressing cell lines, HG-829 significantly enhanced cytotoxicity to daunorubicin, paclitaxel, vinblastine, vincristine, and etoposide. Coadministration of HG-829 fully restored in vivo antitumor activity of daunorubicin in mice without added toxicity. Functional assays showed that HG-829 is not a Pgp substrate or competitive inhibitor of Pgp-mediated drug efflux but rather acts as a noncompetitive modulator of P-glycoprotein transport function. Taken together, our findings indicate that HG-829 is a potent, long-acting, and noncompetitive modulator of P-glycoprotein export function that may offer therapeutic promise for multidrug-resistant malignancies.