IU Indianapolis ScholarWorks :: Browsing by Author "Nalepa, Grzegorz"

Browsing by Author "Nalepa, Grzegorz"

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A Barth Syndrome Patient-Derived D75H Point Mutation in TAFAZZIN Drives Progressive Cardiomyopathy in Mice
(MDPI, 2024-07-27) Snider, Paige L.; Sierra Potchanant, Elizabeth A.; Sun, Zejin; Edwards, Donna M.; Chan, Ka-Kui; Matias, Catalina; Awata, Junya; Sheth, Aditya; Pride, P. Melanie; Payne, R. Mark; Rubart, Michael; Brault, Jeffrey J.; Chin, Michael T.; Nalepa, Grzegorz; Conway, Simon J.; Anatomy, Cell Biology and Physiology, School of Medicine
Cardiomyopathy is the predominant defect in Barth syndrome (BTHS) and is caused by a mutation of the X-linked Tafazzin (TAZ) gene, which encodes an enzyme responsible for remodeling mitochondrial cardiolipin. Despite the known importance of mitochondrial dysfunction in BTHS, how specific TAZ mutations cause diverse BTHS heart phenotypes remains poorly understood. We generated a patient-tailored CRISPR/Cas9 knock-in mouse allele (TazPM) that phenocopies BTHS clinical traits. As TazPM males express a stable mutant protein, we assessed cardiac metabolic dysfunction and mitochondrial changes and identified temporally altered cardioprotective signaling effectors. Specifically, juvenile TazPM males exhibit mild left ventricular dilation in systole but have unaltered fatty acid/amino acid metabolism and normal adenosine triphosphate (ATP). This occurs in concert with a hyperactive p53 pathway, elevation of cardioprotective antioxidant pathways, and induced autophagy-mediated early senescence in juvenile TazPM hearts. However, adult TazPM males exhibit chronic heart failure with reduced growth and ejection fraction, cardiac fibrosis, reduced ATP, and suppressed fatty acid/amino acid metabolism. This biphasic changeover from a mild-to-severe heart phenotype coincides with p53 suppression, downregulation of cardioprotective antioxidant pathways, and the onset of terminal senescence in adult TazPM hearts. Herein, we report a BTHS genotype/phenotype correlation and reveal that absent Taz acyltransferase function is sufficient to drive progressive cardiomyopathy.
An abnormal bone marrow microenvironment contributes to hematopoietic dysfunction in Fanconi anemia
(Ferrata Storti Foundation, 2017-06) Zhou, Yuan; He, Yongzheng; Xing, Wen; Zhang, Peng; Shi, Hui; Chen, Shi; Shi, Jun; Bai, Jie; Rhodes, Steven D.; Zhang, Fengqui; Yuan, Jin; Yang, Xianlin; Zhu, Xiaofan; Li, Yan; Hanenberg, Helmut; Xu, Mingjiang; Robertson, Kent A.; Yuan, Weiping; Nalepa, Grzegorz; Cheng, Tao; Clapp, D. Wade; Yang, Feng-Chun; Pediatrics, School of Medicine
Fanconi anemia is a complex heterogeneous genetic disorder with a high incidence of bone marrow failure, clonal evolution to acute myeloid leukemia and mesenchymal-derived congenital anomalies. Increasing evidence in Fanconi anemia and other genetic disorders points towards an interdependence of skeletal and hematopoietic development, yet the impact of the marrow microenvironment in the pathogenesis of the bone marrow failure in Fanconi anemia remains unclear. Here we demonstrated that mice with double knockout of both Fancc and Fancg genes had decreased bone formation at least partially due to impaired osteoblast differentiation from mesenchymal stem/progenitor cells. Mesenchymal stem/progenitor cells from the double knockout mice showed impaired hematopoietic supportive activity. Mesenchymal stem/progenitor cells of patients with Fanconi anemia exhibited similar cellular deficits, including increased senescence, reduced proliferation, impaired osteoblast differentiation and defective hematopoietic stem/progenitor cell supportive activity. Collectively, these studies provide unique insights into the physiological significance of mesenchymal stem/progenitor cells in supporting the marrow microenvironment, which is potentially of broad relevance in hematopoietic stem cell transplantation.
A child with dyserythropoietic anemia and megakaryocyte dysplasia due to a novel 5′UTR GATA1s splice mutation
(Wiley, 2016-05) Zucker, Jacob; Temm, Constance; Czader, Magdalena; Nalepa, Grzegorz; Department of Pediatrics, IU School of Medicine
We describe a child with dyserythropoietic anemia, thrombocytosis, functional platelet defect, and megakaryocyte dysplasia. We show that (i) this constellation of hematopoietic abnormalities was due to a germline mutation within the 5' untranslated region (5'UTR) of globin transcription factor 1 (GATA1); (ii) the mutation impaired a 5'UTR GATA1 splicing site, with promoted production of the shortened GATA1 isoform lacking the N-terminus; and (iii) expression of the GATA1 N-terminus is restricted to erythroblasts and megakaryocytes in normal marrow, consistent with the patient's abnormal erythropoiesis and megakaryopoiesis. Our findings provide insights into the clinically relevant in vivo function of the N-terminal domain of GATA1 in human hematopoiesis.
Clinical features and outcomes of patients with Shwachman-Diamond syndrome and myelodysplastic syndrome or acute myeloid leukaemia: a multicentre, retrospective, cohort study
(Elsevier, 2020-03) Myers, Kasiani C.; Furutani, Elissa; Weller, Edie; Siegele, Bradford; Galvin, Ashley; Arsenault, Valerie; Alter, Blanche P.; Boulad, Farid; Bueso-Ramos, Carlos; Burroughs, Lauri; Castillo, Paul; Connelly, James; Davies, Stella M.; DiNardo, Courtney D.; Hanif, Iftikhar; Ho, Richard H.; Karras, Nicole; Manalang, Michelle; McReynolds, Lisa J.; Nakano, Taizo A.; Nalepa, Grzegorz; Norkin, Maxim; Oberley, Matthew J.; Orgel, Etan; Pastore, Yves D.; Rosenthal, Joseph; Walkovich, Kelly; Larson, Jordan; Malsch, Maggie; Elghetany, M. Tarek; Fleming, Mark D.; Shimamura, Akiko; Pediatrics, School of Medicine
Background: Data to inform surveillance and treatment for leukaemia predisposition syndromes are scarce and recommendations are largely based on expert opinion. This study aimed to investigate the clinical features and outcomes of patients with myelodysplastic syndrome or acute myeloid leukaemia and Shwachman-Diamond syndrome, an inherited bone marrow failure disorder with high risk of developing myeloid malignancies. Methods: We did a multicentre, retrospective, cohort study in collaboration with the North American Shwachman-Diamond Syndrome Registry. We reviewed patient medical records from 17 centres in the USA and Canada. Patients with a genetic (biallelic mutations in the SBDS gene) or clinical diagnosis (cytopenias and pancreatic dysfunction) of Shwachman-Diamond syndrome who developed myelodysplastic syndrome or acute myeloid leukaemia were eligible without additional restriction. Medical records were reviewed between March 1, 2001, and Oct 5, 2017. Masked central review of bone marrow pathology was done if available to confirm leukaemia or myelodysplastic syndrome diagnosis. We describe the clinical features and overall survival of these patients. Findings: We initially identified 37 patients with Shwachman-Diamond syndrome and myelodysplastic syndrome or acute myeloid leukaemia. 27 patients had samples available for central pathology review and were reclassified accordingly (central diagnosis concurred with local in 15 [56%] cases), 10 had no samples available and were classified based on the local review data, and 1 patient was excluded at this stage as not eligible. 36 patients were included in the analysis, of whom 10 (28%) initially presented with acute myeloid leukaemia and 26 (72%) initially presented with myelodysplastic syndrome. With a median follow-up of 4·9 years (IQR 3·9-8·4), median overall survival for patients with myelodysplastic syndrome was 7·7 years (95% CI 0·8-not reached) and 0·99 years (95% CI 0·2-2·4) for patients with acute myeloid leukaemia. Overall survival at 3 years was 11% (95% CI 1-39) for patients with leukaemia and 51% (29-68) for patients with myelodysplastic syndrome. Management and surveillance were variable. 18 (69%) of 26 patients with myelodysplastic syndrome received upfront therapy (14 haematopoietic stem cell transplantation and 4 chemotherapy), 4 (15%) patients received no treatment, 2 (8%) had unavailable data, and 2 (8%) progressed to acute myeloid leukaemia before receiving treatment. 12 patients received treatment for acute myeloid leukaemia-including the two patients initially diagnosed with myelodysplastic who progressed- two (16%) received HSCT as initial therapy and ten (83%) received chemotherapy with intent to proceed with HSCT. 33 (92%) of 36 patients (eight of ten with leukaemia and 25 of 26 with myelodysplastic syndrome) were known to have Shwachman-Diamond syndrome before development of a myeloid malignancy and could have been monitored with bone marrow surveillance. Bone marrow surveillance before myeloid malignancy diagnosis was done in three (33%) of nine patients with leukaemia for whom surveillance status was confirmed and 11 (46%) of 24 patients with myelodysplastic syndrome. Patients monitored had a 3-year overall survival of 62% (95% CI 32-82; n=14) compared with 28% (95% CI 10-50; n=19; p=0·13) without surveillance. Six (40%) of 15 patients with available longitudinal data developed myelodysplastic syndrome in the setting of stable blood counts. Interpretation: Our results suggest that prognosis is poor for patients with Shwachman-Diamond syndrome and myelodysplastic syndrome or acute myeloid leukaemia owing to both therapy-resistant disease and treatment-related toxicities. Improved surveillance algorithms and risk stratification tools, studies of clonal evolution, and prospective trials are needed to inform effective prevention and treatment strategies for leukaemia predisposition in patients with Shwachman-Diamond syndrome.
A Critical Role of CDKN3 in Bcr-Abl-Mediated Tumorigenesis
(PLoS, 2014-10) Chen, Qinghuang; Chen, Ke; Guo, Guijie; Li, Fang; Chen, Chao; Wang, Song; Nalepa, Grzegorz; Huang, Shile; Chen, Ji-Long; Department of Medical and Molecular Genetics, IU School of Medicine
CDKN3 (cyclin-dependent kinase inhibitor 3), a dual specificity protein phosphatase, dephosphorylates cyclin-dependent kinases (CDKs) and thus functions as a key negative regulator of cell cycle progression. Deregulation or mutations of CDNK3 have been implicated in various cancers. However, the role of CDKN3 in Bcr-Abl-mediated chronic myelogenous leukemia (CML) remains unknown. Here we found that CDKN3 acts as a tumor suppressor in Bcr-Abl-mediated leukemogenesis. Overexpression of CDKN3 sensitized the K562 leukemic cells to imanitib-induced apoptosis and dramatically inhibited K562 xenografted tumor growth in nude mouse model. Ectopic expression of CDKN3 significantly reduced the efficiency of Bcr-Abl-mediated transformation of FDCP1 cells to growth factor independence. In contrast, depletion of CDKN3 expression conferred resistance to imatinib-induced apoptosis in the leukemic cells and accelerated the growth of xenograph leukemia in mice. In addition, we found that CDKN3 mutant (CDKN3-C140S) devoid of the phosphatase activity failed to affect the K562 leukemic cell survival and xenografted tumor growth, suggesting that the phosphatase of CDKN3 was required for its tumor suppressor function. Furthermore, we observed that overexpression of CDKN3 reduced the leukemic cell survival by dephosphorylating CDK2, thereby inhibiting CDK2-dependent XIAP expression. Moreover, overexpression of CDKN3 delayed G1/S transition in K562 leukemic cells. Our results highlight the importance of CDKN3 in Bcr-Abl-mediated leukemogenesis, and provide new insights into diagnostics and therapeutics of the leukemia.
Cyclin E-CDK2 Protein Phosphorylates Plant Homeodomain Finger Protein 8 (PHF8) and Regulates Its Function in the Cell Cycle
(2015-02) Sun, Liping; Huang, Yan; Wei, Qian; Tong, Xiaomei; Cai, Rong; Nalepa, Grzegorz; Ye, Xin; Department of Pediatrics, Indiana University School of Medicine
Cyclin E-CDK2 is a key regulator in G1/S transition. Previously, we identified a number of CDK2-interacting proteins, including PHF8 (plant homeodomain finger protein 8). In this report, we confirmed that PHF8 is a novel cyclin E-CDK2 substrate. By taking the approach of mass spectrometry, we identified that PHF8 Ser-844 is phosphorylated by cyclin E-CDK2. Immunoblotting analysis indicated that WT PHF8 demethylates histone H3K9me2 more efficiently than the cyclin E-CDK2 phosphorylation-deficient PHF8-S844A mutant. Furthermore, flow cytometry analysis showed that WT PHF8 promotes S phase progression more robustly than PHF8-S844A. Real-time PCR results demonstrated that PHF8 increases transcription of cyclin E, E2F3, and E2F7 to significantly higher levels compared with PHF8-S844A. Further analysis by ChIP assay indicated that PHF8 binds to the cyclin E promoter stronger than PHF8-S844A and reduces the H3K9me2 level at the cyclin E promoter more efficiently than PHF8-S844A. In addition, we found that cyclin E-CDK2-mediated phosphorylation of PHF8 Ser-844 promotes PHF8-dependent rRNA transcription in luciferase reporter assays and real-time PCR. Taken together, these results indicate that cyclin E-CDK2 phosphorylates PHF8 to stimulate its demethylase activity to promote rRNA transcription and cell cycle progression.
Evaluation and Management of Chronic Pancytopenia
(AAP, 2016-03) Sharma, Richa; Nalepa, Grzegorz; Department of Pediatrics, IU School of Medicine
Diagnostic evaluation and management of children with chronic pancytopenia is highly complex. (1)(2)(3) Clinicians should be aware of the subtle signs and symptoms of acquired and congenital childhood pancytopenias to maintain a high index of suspicion, streamline the referral process, and function within their patients’ multidisciplinary medical homes.
FANCA maintains genomic stability through regulating BUBR1 acetylation
(2017-08) Abdul Sater, Zahi Abass; Nalepa, Grzegorz; Clapp, Wade; Goebl, Mark; Wek, Ronald
Fanconi Anemia (FA), a chromosomal instability syndrome, is characterized by bone marrow failure, genetic malformations, and predisposition to malignancies like acute myeloid leukemia (AML) and solid tumors. FA is caused by germline bi-allelic mutations in one of 21 known FA pathway genes and somatic mutations in FA genes are also found in a variety of sporadic cancers. Recently, numerous reports have discovered that the protective function of the FA pathway extends beyond its canonical role in regulation of DNA repair in interphase. In particular, the FA pathway has been shown to function in essential mitotic processes including spindle assembly checkpoint (SAC), cytokinesis, and centrosome maintenance. Understanding of the mechanistic origins of genomic instability leading to carcinogenesis and bone marrow failure has important scientific and clinical implications. To this end, using a micronucleus assay, we showed that both interphase DNA damage and mitotic errors contribute to genomic instability in FA ex vivo and in vivo. Functional studies of primary FA patient cells coupled with super-resolution microscopy revealed that FANCA is important for centrosome dependent spindle assembly supporting the protective role of FA pathway in mitotic processes. Furthermore, we dissected the interactions between the FA pathway and cellular kinase networks by employing a synthetic lethality sh-RNA screen targeting all human kinases. We mapped kinases that were synthetically lethal upon loss of FANCA, particularly those involved in highly conserved signal transduction pathways governing proliferation and cell cycle homeostasis. We mechanistically show that loss of FANCA, the most abundant FA subtype, results in in premature degradation of the mitotic kinase BUBR1 and faster mitotic exit. We further demonstrate that FANCA is important for PCAF-dependent acetylation of BUBR1 to prevent its premature degradation. Our results deepen our understanding of the molecular functions of the FA pathway in mitosis and uncover a mechanistic connection between FANCA and SAC phosphosignaling networks. These findings support the notion that further weakening the SAC through targeting kinases like BUBR1 in FA-deficient cancers may prove to be a rational therapeutic strategy.
FANCA safeguards interphase and mitosis during hematopoiesis in vivo
(Elsevier, 2015-12) Abdul-Sater, Zahi; Cerabona, Donna; Potchanant, Elizabeth Sierra; Sun, Zejin; Enzor, Rikki; He, Ying; Robertson, Kent; Goebel, W. Scott; Nalepa, Grzegorz; Department of Pediatrics, IU School of Medicine
The Fanconi anemia (FA/BRCA) signaling network controls multiple genome-housekeeping checkpoints, from interphase DNA repair to mitosis. The in vivo role of abnormal cell division in FA remains unknown. Here, we quantified the origins of genomic instability in FA patients and mice in vivo and ex vivo. We found that both mitotic errors and interphase DNA damage significantly contribute to genomic instability during FA-deficient hematopoiesis and in nonhematopoietic human and murine FA primary cells. Super-resolution microscopy coupled with functional assays revealed that FANCA shuttles to the pericentriolar material to regulate spindle assembly at mitotic entry. Loss of FA signaling rendered cells hypersensitive to spindle chemotherapeutics and allowed escape from the chemotherapy-induced spindle assembly checkpoint. In support of these findings, direct comparison of DNA crosslinking and anti-mitotic chemotherapeutics in primary FANCA-/- cells revealed genomic instability originating through divergent cell cycle checkpoint aberrations. Our data indicate that FA/BRCA signaling functions as an in vivo gatekeeper of genomic integrity throughout interphase and mitosis, which may have implications for future targeted therapies in FA and FA-deficient cancers.
Fancc regulates the spindle assembly checkpoint to prevent tumorigenesis in vivo
(2017-06) Edwards, Donna Marie; Clapp, D. Wade; Nalepa, Grzegorz; Harrington, Maureen A.; Goebl, Mark G.
The Fanconi anemia (FA) pathway consists of 21 genes that maintain genomic stability and prevent cancer. Biallelic mutations within this network cause Fanconi anemia, an inherited bone marrow failure and cancer predisposition syndrome. Heterozygous inborn mutations in FA genes increase risk of breast/ovarian cancers, and somatic mutations occur in malignancies in non-Fanconi patients. Understanding the tumor suppressive functions of FA signaling is important for the study of Fanconi anemia, inherited cancers, and sporadic cancers. The FA network functions as a genome guardian throughout the cell cycle. In addition to the well-established roles of FA proteins in interphase DNA replication/repair, the FA pathway controls mitosis by regulating the spindle assembly checkpoint (SAC) to ensure proper chromosome segregation. The SAC consists of several tumor suppressors, including Mad2, and SAC impairment predisposes to aneuploidy and cancer. However, the in vivo contribution of SAC dysfunction to malignant transformation of FA-deficient cells remains unknown. Furthermore, the mechanisms by which FA proteins regulate the SAC are unclear. To test whether SAC dysfunction drives genomic instability and tumorigenesis in FA, we generated a novel FA-SAC model by intercrossing Fancc-/- and Mad2+/- mice. The intercrossed mice displayed heightened aneuploidy secondary to exacerbated SAC dysfunction. Importantly, these mice were prone to developing hematologic malignancies, particularly leukemia, faithfully recapitulating the clinical phenotype of Fanconi anemia. Upon establishing SAC dysfunction as a driver of tumorigenesis in FA, we next explored the mechanism by which FANCC regulates the SAC. We demonstrated that the mitotic kinase CDK1 phosphorylates FANCC to regulate subcellular localization and SAC function of FANCC during mitosis. Our study highlights the essential role of compromised chromosome segregation in the development of leukemia due to impaired FA signaling. This work furthers our knowledge of FANCC signaling at the SAC, and has implications for future use of mitotic-centered therapies for FA-associated tumors.

Browsing by Author "Nalepa, Grzegorz"

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