Overexpression of stathmin in oral squamous-cell carcinoma: correlation with tumour progression and poor prognosis

Stathmin is an intracellular phosphoprotein that is overexpressed in a number of human malignancies. Our previous study using proteomic profiling showed that significant upregulation of stathmin occurs in oral squamous-cell carcinoma (OSCC)-derived cell lines. In the current study, to determine the potential involvement of stathmin in OSCC, we evaluated the state of stathmin protein and mRNA expression in OSCC-derived cell lines and human primary OSCCs. A significant increase in stathmin expression was observed in all OSCC-derived cell lines examined compared to human normal oral keratinocytes. In immunohistochemistry, 65% of the OSCCs were positive for stathmin, and no immunoreaction was observed in corresponding normal tissues. Real-time quantitative reverse transcriptase–polymerase chain reaction data were consistent with the protein expression status. Moreover, stathmin expression status was correlated with the TNM stage grading. Furthermore, we found a statistical correlation between the protein expression status and disease-free survival (P=0.029). These results suggest that expression of stathmin could contribute to cancer progression/prognosis, and that stathmin may have potential as a biomarker and a therapeutic target for OSCC

Epigenetic abnormalities in myeloproliferative neoplasms: a target for novel therapeutic strategies

The myeloproliferative neoplasms (MPNs) are a group of clonal hematological malignancies characterized by a hypercellular bone marrow and a tendency to develop thrombotic complications and to evolve to myelofibrosis and acute leukemia. Unlike chronic myelogenous leukemia, where a single disease-initiating genetic event has been identified, a more complicated series of genetic mutations appear to be responsible for the BCR-ABL1-negative MPNs which include polycythemia vera, essential thrombocythemia, and primary myelofibrosis. Recent studies have revealed a number of epigenetic alterations that also likely contribute to disease pathogenesis and determine clinical outcome. Increasing evidence indicates that alterations in DNA methylation, histone modification, and microRNA expression patterns can collectively influence gene expression and potentially contribute to MPN pathogenesis. Examples include mutations in genes encoding proteins that modify chromatin structure (EZH2, ASXL1, IDH1/2, JAK2V617F, and IKZF1) as well as epigenetic modification of genes critical for cell proliferation and survival (suppressors of cytokine signaling, polycythemia rubra vera-1, CXC chemokine receptor 4, and histone deacetylase (HDAC)). These epigenetic lesions serve as novel targets for experimental therapeutic interventions. Clinical trials are currently underway evaluating HDAC inhibitors and DNA methyltransferase inhibitors for the treatment of patients with MPNs

The role of stathmin, a regulator of mitosis, in hematopoiesis

Steel, JC ORCiD: 0000-0003-3608-7542Introduction Stathmin is a 17KDa cytosolic protein that plays an important role in the regulation of microtubule dynamics, mitotic spindle formation, cell cycle progression and cell differentiation. Stathmin knockout (KO) mice were initially reported to have a normal phenotype but were subsequently shown to develop an age-related neurological phenotype with axonopathy evident in both central and peripheral nervous systems. These mice were also shown to have a defect in recovery from acute ischemic renal injury. We had previously shown that stathmin plays an important role in the differentiation and proliferation of megakaryocytes (MK) and that down-regulation of stathmin is necessary for the maturation of MK and platelet production in vitro. In this study, we investigated the role of stathmin in megakaryopoiesis and hematopoiesis in vivo using the stathmin KO mouse as an experimental model. Results Stathmin KO mice had lower platelet (PLT) counts at 3 weeks of age when compared to WT mice. The WT mice had a mean PLT count of 662 ± 27 K/μL while KO mice had a mean PLT count of 543 ± 37 K/μL. This correlated with larger and fewer MK in the bone marrow of KO mice (WT: 4.2 ± 0.7 MK/40X field; KO: 3.6 ± 0.2 MK/40X field). Furthermore, in the spleen, there was a 10 fold decrease in the number of MK in KO mice compared to WT mice (6.6 ± 0.6 vs 0.7 ± 0.1 MK/40X field). By 8 weeks, PLT counts and MK size and numbers in the bone marrow and spleen were similar in WT and KO mice. Interestingly, by 16 weeks, the mean PLT of KO mice became significantly higher than that of WT and by 40 weeks, the mean PLT count of KO mice was 1379 ± 100K/μL compared to 1045 ± 120K/μL in WT mice (P<0.05). Microscopic analysis of the bone marrow at 46 weeks of age showed approximately 50% more MK in KO mice compared to WT mice. Differences in red blood cell counts (RBC) were also observed. While at 3 weeks, there were no significant differences between the 2 groups, at 8 weeks, KO mice had significantly lower RBC counts, hemoglobin levels (Hb) and hematocrit (HCT). This trend continued until the last measurement recorded at 40 weeks. Mean RBC in WT mice was 10.5 ± 0.1M/μL compared to 8.9 ± 0.2M/μL in KO mice. The mean corpuscular volume (MCV) and the red blood cell distribution width (RDW) were consistently higher in KO mice than in WT mice. No significant differences were noted in white blood cell counts. Bone marrow cell counts were significantly lower in KO mice when compared to WT mice at different ages from 3–40 weeks. Progenitor cell assays from 10–12 week old animals have shown that bone marrow from KO mice produce significantly fewer BFU-E and Pre-B colonies while no differences were observed in CFU-GMs. Conclusions The phenotypic characteristics of stathmin KO mice confirmed our prior in vitro findings that suggested a role for stathmin in megakaryopoiesis. We expected to see a decrease in the number of platelets and MK coupled with an increase in MK size. This was confirmed in stathmin KO mice at 3 weeks of age. However, we did not expect to see the marked increase in the number of platelets and MK that was observed as the mice aged. The exact mechanism for this has not been identified. Interestingly, the stathmin KO mice exhibited characteristic features of megaloblastic anemia including mild anemia and a significant increase in MCV and RDW. The megaloblastic anemia that is seen in the presence of B12 and folate deficiency results from interference with DNA synthesis resulting in asynchronous maturation of the nucleus and the cytoplasm. We believe a similar phenomenon is occurring in the stathmin KO mice. The deficiency of stathmin results in aberrant exit from mitosis, thereby delaying nuclear maturation and resulting in the megaloblastic features. Thus, the deficiency of stathmin in the KO mice results in two hematopoietic phenotypes that are seen in humans, megaloblastic anemia and thrombocytosis. It is unclear whether mutations of stathmin in humans might result in similar phenotypes. This is a question that will require further investigation. Future studies will investigate the compensatory mechanisms that result in the switch from decreased to increased platelet production as the mice age. Furthermore, examining the effects of hematopoietic stress (e.g. response to chemotherapy or bleeding) in stathmin KO mice might also elucidate a role for stathmin in the recovery from hematopoietic injury as was seen in acute ischemic renal injury

The role of stathmin, a regulator of mitosis, in hematopoiesis

Introduction Stathmin is a 17KDa cytosolic protein that plays an important role in the regulation of microtubule dynamics, mitotic spindle formation, cell cycle progression and cell differentiation. Stathmin knockout (KO) mice were initially reported to have a normal phenotype but were subsequently shown to develop an age-related neurological phenotype with axonopathy evident in both central and peripheral nervous systems. These mice were also shown to have a defect in recovery from acute ischemic renal injury. We had previously shown that stathmin plays an important role in the differentiation and proliferation of megakaryocytes (MK) and that down-regulation of stathmin is necessary for the maturation of MK and platelet production in vitro. In this study, we investigated the role of stathmin in megakaryopoiesis and hematopoiesis in vivo using the stathmin KO mouse as an experimental model. Results Stathmin KO mice had lower platelet (PLT) counts at 3 weeks of age when compared to WT mice. The WT mice had a mean PLT count of 662 ± 27 K/μL while KO mice had a mean PLT count of 543 ± 37 K/μL. This correlated with larger and fewer MK in the bone marrow of KO mice (WT: 4.2 ± 0.7 MK/40X field; KO: 3.6 ± 0.2 MK/40X field). Furthermore, in the spleen, there was a 10 fold decrease in the number of MK in KO mice compared to WT mice (6.6 ± 0.6 vs 0.7 ± 0.1 MK/40X field). By 8 weeks, PLT counts and MK size and numbers in the bone marrow and spleen were similar in WT and KO mice. Interestingly, by 16 weeks, the mean PLT of KO mice became significantly higher than that of WT and by 40 weeks, the mean PLT count of KO mice was 1379 ± 100K/μL compared to 1045 ± 120K/μL in WT mice (P<0.05). Microscopic analysis of the bone marrow at 46 weeks of age showed approximately 50% more MK in KO mice compared to WT mice. Differences in red blood cell counts (RBC) were also observed. While at 3 weeks, there were no significant differences between the 2 groups, at 8 weeks, KO mice had significantly lower RBC counts, hemoglobin levels (Hb) and hematocrit (HCT). This trend continued until the last measurement recorded at 40 weeks. Mean RBC in WT mice was 10.5 ± 0.1M/μL compared to 8.9 ± 0.2M/μL in KO mice. The mean corpuscular volume (MCV) and the red blood cell distribution width (RDW) were consistently higher in KO mice than in WT mice. No significant differences were noted in white blood cell counts. Bone marrow cell counts were significantly lower in KO mice when compared to WT mice at different ages from 3–40 weeks. Progenitor cell assays from 10–12 week old animals have shown that bone marrow from KO mice produce significantly fewer BFU-E and Pre-B colonies while no differences were observed in CFU-GMs. Conclusions The phenotypic characteristics of stathmin KO mice confirmed our prior in vitro findings that suggested a role for stathmin in megakaryopoiesis. We expected to see a decrease in the number of platelets and MK coupled with an increase in MK size. This was confirmed in stathmin KO mice at 3 weeks of age. However, we did not expect to see the marked increase in the number of platelets and MK that was observed as the mice aged. The exact mechanism for this has not been identified. Interestingly, the stathmin KO mice exhibited characteristic features of megaloblastic anemia including mild anemia and a significant increase in MCV and RDW. The megaloblastic anemia that is seen in the presence of B12 and folate deficiency results from interference with DNA synthesis resulting in asynchronous maturation of the nucleus and the cytoplasm. We believe a similar phenomenon is occurring in the stathmin KO mice. The deficiency of stathmin results in aberrant exit from mitosis, thereby delaying nuclear maturation and resulting in the megaloblastic features. Thus, the deficiency of stathmin in the KO mice results in two hematopoietic phenotypes that are seen in humans, megaloblastic anemia and thrombocytosis. It is unclear whether mutations of stathmin in humans might result in similar phenotypes. This is a question that will require further investigation. Future studies will investigate the compensatory mechanisms that result in the switch from decreased to increased platelet production as the mice age. Furthermore, examining the effects of hematopoietic stress (e.g. response to chemotherapy or bleeding) in stathmin KO mice might also elucidate a role for stathmin in the recovery from hematopoietic injury as was seen in acute ischemic renal injury

Preclinical rationale for TGF-\u3b2 inhibition as a therapeutic target for the treatment of myelofibrosis

To assess the role of abnormal transforming growth factor-beta (TGF-\u3b2) signaling in the pathogenesis of primary myelofibrosis (PMF), the effects of the TGF-\u3b2 receptor-1 kinase inhibitor SB431542 on ex&nbsp;vivo expansion of hematopoietic cells in cultures from patients with JAK2V617+-polycythemia vera (PV) or PMF (JAK2V617F+, CALRpQ365f+, or unknown) and from normal sources (adult blood, AB, or cord blood, CB) were compared. In cultures of normal sources, SB431542 significantly increased by 2.5-fold the number of progenitor cells generated by days 1\u20132 (CD34+) and 6 (colony-forming cells) (CB) and that of precursor cells, mostly immature erythroblasts, by days 14\u201317 (AB and CB). In cultures of JAK2V617F+-PV, SB431542 increased by twofold the numbers of progenitor cells by day 10 and had no effect on that of precursors cells by days 12\u201317 ( 3cfourfold increase in all cases). In contrast, SB431542 had no effect on the number of either progenitor or precursor cells in cultures of JAK2V617F+ and CALR pQ365fs+ PMF. These ontogenetic- and disease-specific effects were associated with variegation in the ability of SB431542 to induce CD34+ cells from AB (increased), CB (decreased), or PV and PMF (unaffected) into cycle and erythroblasts in proliferation (increased for AB and PV and unaffected for CB and PMF). Differences in expansion of erythroblasts from AB, CB, and PV were associated with differences in activation of TGF-\u3b2 signaling (SHCY317, SMAD2S245/250/255, and SMAD1S/S/SMAD5S/S/SMAD8S/S) detectable in these cells by phosphoproteomic profiling. In conclusion, treatment with TGF-\u3b2 receptor-1 kinase inhibitors may reactivate normal hematopoiesis in PMF patients, providing a proliferative advantage over the unresponsive malignant clone

Development of an optimized closed and semi-automatic protocol for Good Manufacturing Practice manufacturing of tumor-infiltrating lymphocytes in a hospital environment.

Several studies report on Good Manufacturing Process (GMP)-compliant manufacturing protocols for the ex vivo expansion of tumor-infiltrating lymphocytes (TILs) for the treatment of patients with refractory melanoma and other solid malignancies. Further opportunities for improvements in terms of ergonomy and operating time have been identified. To enable GMP-compliant TILs production for adoptive cell therapy needs, a simple automated and reproducible protocol for TILs manufacturing with the use of a closed system was developed and implemented at the authors' institution. This protocol enabled significant operating time reduction during TILs expansion while allowing the generation of high-quality TILs products. A simplified and efficient method of TILs expansion will enable the broadening of individualized tumor therapy and will increase patients' access to state-of-the-art TILs adoptive cell therapy treatment

Design of embedded chimeric peptide nucleic acids that efficiently enter and accurately reactivate gene expression in vivo

Pharmacological treatments designed to reactivate fetal γ-globin can lead to an effective and successful clinical outcome in patients with hemoglobinopathies. However, new approaches remain highly desired because such treatments are not equally effective for all patients, and toxicity issues remain. We have taken a systematic approach to develop an embedded chimeric peptide nucleic acid (PNA) that effectively enters the cell and the nucleus, binds to its target site at the human fetal γ-globin promoter, and reactivates this transcript in adult transgenic mouse bone marrow and human primary peripheral blood cells. In vitro and in vivo DNA-binding assays in conjunction with live-cell imaging have been used to establish and optimize chimeric PNA design parameters that lead to successful gene activation. Our final molecule contains a specific γ-promoter-binding PNA sequence embedded within two amino acid motifs: one leads to efficient cell/nuclear entry, and the other generates transcriptional reactivation of the target. These embedded PNAs overcome previous limitations and are generally applicable to the design of in vivo transcriptional activation reagents that can be directed to any promoter region of interest and are of direct relevance to clinical applications that would benefit from such a need