Degree Granting Department
Cell Biology, Microbiology, and Molecular Biology
Alan F. List
Erythropoietin, GTPases, Lipid Rafts, RNF41
Myelodysplastic syndromes (MDS) include a spectrum of stem cell malignancies characterized by ineffective hematopoiesis and predisposition to acute myeloid leukemia (AML) transformation. Patients are predominantly older (greater than 60 years old), with progressive cytopenias resulting from ineffective and cytologically dysplastic hematopoiesis. MDS subtypes are classified by morphologic features and bone marrow blast percentage, as well as cytogenetic pattern, as is the case for deletion 5q MDS. Interstitial deletion of the long arm of chromosome 5, del(5q), is the most common chromosomal abnormality in patients with MDS, and the 5q- syndrome, represents a distinct subset of del(5q) MDS characterized by an isolated deletion, megakaryocyte dysplasia, hypoplastic anemia, and an indolent natural history. MDS risk stratification is most commonly based on the International Prognostic Scoring System (IPSS) with survival outcomes ranging from a few months to many years based on risk factors. There are several therapeutic options for MDS including hematopoietic growth factors, immunosuppressive therapy, azanucleosides, and allogeneic stem cell transplant, however, there is still a need for more effective treatment options, particularly targeted therapeutics. One of the most effective treatments for MDS is selective for del(5q) MDS, and is the second generation immunomodulatory agent, lenalidomide (LEN).
LEN is an analog of the known teratogen, thalidomide, and has broad biological effects including selective cytotoxicity to del(5q) clones, activation of T-cells, and expansion of erythroid precursors. In patients with del(5q) MDS, LEN is effective in up to 75% of patients, however, 50% of patients will become resistant within 2-3 years of treatment response. Studies in normal hematopoietic progenitors have shown that LEN induces expansion of the primitive erythroid precursors, which our laboratory has shown is accompanied by sensitization of progenitors to ligand induced erythropoietin receptor (EpoR) signaling. This sensitization is evidenced by increased and prolonged activation of the Signal Transducer and Activator of Transcription 5 (STAT5), compared to Epo stimulation alone. Although EpoR signaling is augmented by LEN, the exact mechanisms by which this is mediated to result in erythroid expansion are not fully characterized. In del(5q) MDS, we have shown that LEN selectively suppresses del(5q) clones via inhibition of the haploinsufficient phosphatases Cdc25c and PP2a, as well as stabilizing the human homolog of the murine double minute-2 protein (MDM2) to decrease expression of the tumor suppressor, p53, however, the mechanisms of action of LEN in non-del(5q) MDS remains elusive.
Although most anemic MDS patients have normal or elevated endogenous levels of Epo, as well as comparable levels of progenitor EpoR density relative to healthy individuals, the biologic pathology underlying the impaired EpoR signaling in MDS is poorly defined. Recent reports have shown that membrane microdomains are important for T-cell, c-kit, and integrin signaling, however, there have been no reports on EpoR membrane localization. Lipid rafts are discrete membrane entities that provide platforms by which receptors aggregate and initiate downstream signaling. Furthermore, reports have indicated that there is a decrease in lipid raft density in GM-CSF primed MDS neutrophils, that consequently impaired production of reactive oxygen species (ROS) after fMLP stimulation, suggesting a role of rafts in MDS disease biology. Based on the role of rafts in signaling, and potential role in MDS pathogenesis, we sought to determine whether there was specific membrane localization of EpoR to the raft fractions, and whether disruption of rafts in MDS erythroids could impair EpoR signaling. To address this, we first examined the membrane localization of EpoR on the cell surface. We show here that EpoR translocates to lipid rafts in both erythroid progenitor cell lines as well as primary progenitor cells after stimulation by Epo. Furthermore, we found that Epo stimulation increases the assembly of lipid rafts, as well as the aggregation of rafts on the cell surface. Epo stimulation not only promoted the recruitment of EpoR into the raft fractions, but also downstream signaling intermediates such as Janus kinase 2 (Jak2), STAT5, and Lyn kinase. Moreover, a negative regulator of EpoR signaling, the CD45 tyrosine phosphatase, was redistributed outside of raft fractions after Epo stimulation, potentially enhancing receptor signal competence. Furthermore, disruption of lipid rafts by depletion of membrane cholesterol with MâCD (methyl-β-cyclodextrin) inhibited EpoR signaling in both cell lines and primary bone marrow progenitor cells. Additionally, we found that inhibition of Rho-associated, coiled-coil containing protein kinase (ROCK) and/or Ras-related C3 botulinium toxin substrate 1 (Rac1), blocked the recruitment of the receptor into the raft fractions indicating a critical role of these GTPases, and associated proteins, in the transport and localization of EpoR into raft microdomains.
We next asked whether LEN could alter lipid raft assembly in erythroid precursors in the absence of Epo. LEN not only induced raft formation and aggregation but also increased F-actin polymerization. Similar to Epo stimulation, LEN alone was able to induce the recruitment of EpoR, Jak2, and STAT5 into raft fractions. Additionally, CD45 was redistributed outside of raft fractions after LEN treatment. Similarly, inhibition of ROCK blocked LEN induced raft formation and F-actin polymerization, indicating that LEN utilized effectors shared by Epo. Furthermore, LEN was able to increase raft density in raft deficient primary MDS erythroid progenitors. These data demonstrate that LEN may enhance erythroid expansion via induction of EpoR signaling competent raft platforms, to enhance survival and differentiation transcriptional response.
Recently, ribosomal protein (RP), S-14, gene (RPS14) haplodeficiency was found to be a key determinant of the hypoplastic anemia in del(5q) MDS. Allelic loss of RPS14 compromises ribosome assembly, thereby causing nucleolar stress and release of free RPs that bind to and promote the degradation of MDM2, the principal negative regulator of p53. As a result, the accumulation of RPs causes lineage restricted stabilization of p53 in erythroid precursors. Our laboratory and colleagues confirmed that cellular p53 expression levels were elevated in del(5q) erythroid precursors, and that LEN decreased expression in responding patients. However, at the time of LEN treatment failure, p53 expression was again elevated at levels exceeding those at baseline. These results suggest that LEN is initially able to reverse p53 accumulation levels and that this action may be a mechanism by which LEN is selectively cytotoxic to del(5q) clones. Subsequent studies showed that LEN inhibits the cereblon E3 ubiquitin ligase complex, the newly discovered target of LEN. Cereblon has been reported to be the principal protein involved in thalidomide induced teratogenicity. Furthermore, the cytotoxic activity of LEN in multiple myeloma is dependent on cereblon. Our laboratory found that LEN inhibits the auto-ubiquitination of MDM2, thereby stabilizing the protein, and promoting ubiquitination of and ultimately the degradation of p53. Additionally, we found that LEN blocked the binding of free ribosomal proteins to MDM2, which are liberated from the nucleosome by ribosomal stress from RPS14 haploinsufficiency, consequently stabilizing the E3-ubiquitin ligase and fostering p53 degradation.
In non-del(5q) MDS there is no cytotoxicity of MDS clones by LEN, suggesting an alternative method of erythropoiesis rescue. Although we know that LEN promotes the formation of signaling platforms, and recruitment of EpoR, we wished to determine whether there was an effect of LEN on EpoR expression, as EpoR expression is controlled through ubiquitination and proteasomal degradation. Treatment of erythroid progenitor cell lines and primary erythroid precursors with LEN increased cellular expression of Jak2-associated EpoR in a concentration dependent manner. There was no change in mRNA expression, supporting a post transcriptional mechanism. We then investigated whether receptor up-regulation was limited to EpoR, or included other cytokine receptors. We found that LEN induced expression of another Jak2 associated Type I receptor, IL3-R, but did not alter cellular expression of c-kit, a Type II cytokine receptor. Because Type I cytokine receptor turnover is regulated by a shared E3-ubiquitin ligase, and LEN inhibited both MDM2 and cereblon, we evaluated the effects of LEN on the E3-ubiquitin ligase, Ring Finger Protein-41 (RNF41), which regulates steady state or ligand independent, Jak2 associated Type I receptor internalization. We found that LEN inhibited the ubiquitination activity of RNF41, ultimately stabilizing EpoR membrane residence and increasing expression.
In summary, MDS patients display ineffective hematopoiesis likely in part to decreased lipid raft assembly. Stimulation by Epo, or treatment by LEN, not only induced raft formation, but also induced the recruitment of both growth factor receptor, and downstream signaling intermediates into raft fractions to enhance EpoR signal fidelity. We have shown here two methods by which LEN may augment EpoR signaling. First, LEN increases lipid rafts and promotes recruitment of signaling effectors. Second, LEN increases and stabilizes the expression of EpoR through inhibition of the E3 ubiquitin ligase, RNF41. Therefore, we suggest here that LEN may have broad E3 ubiquitin ligase inhibitory effects. These data also indicate that lipid raft upregulation by LEN is mediated through GTPases, suggesting that GTPase activation may also occur via inhibition of specific E3 ubiquitin ligases, a question to be addressed in future studies.
Scholar Commons Citation
Mcgraw, Kathy Lynn, "Interrogation of EpoR Fidelity in Myelodysplastic Syndrome Hematopoiesis and Stabilization by the Immunomodulatory Agent, Lenalidomide" (2013). Graduate Theses and Dissertations.