Current chemotherapies for T cell acute lymphoblastic leukemia (T-ALL) efficiently reduce tumor mass. Nonetheless, disease relapse attributed to survival of preleukemic stem cells (pre-LSCs) is associated with poor prognosis. Herein, we provide direct evidence that pre-LSCs are much less chemosensitive to existing chemotherapy drugs than leukemic blasts because of a distinctive lower proliferative state. Improving therapies for T-ALL requires the development of strategies to target pre-LSCs that are absolutely dependent on their microenvironment. Therefore, we designed a robust protocol for high-throughput screening of compounds that target primary pre-LSCs maintained in a niche-like environment, on stromal cells that were engineered for optimal NOTCH1 activation. The multiparametric readout takes into account the intrinsic complexity of primary cells in order to specifically monitor pre-LSCs, which were induced here by the
Bastien Gerby, Diogo F.T. Veiga, Jana Krosl, Sami Nourreddine, Julianne Ouellette, André Haman, Geneviève Lavoie, Iman Fares, Mathieu Tremblay, Véronique Litalien, Elizabeth Ottoni, Milena Kosic, Dominique Geoffrion, Joël Ryan, Paul S. Maddox, Jalila Chagraoui, Anne Marinier, Josée Hébert, Guy Sauvageau, Benjamin H. Kwok, Philippe P. Roux, Trang Hoang
Certain secretory proteins are known to be critical for maintaining the stemness of stem cells through autocrine signaling. However, the processes underlying the biogenesis, maturation, and secretion of these proteins remain largely unknown. Here we demonstrate that many secretory proteins produced by hematopoietic stem cells (HSCs) undergo exosomal maturation and release that is controlled by vacuolar protein sorting protein 33b (VPS33B). Deletion of
Hao Gu, Chiqi Chen, Xiaoxin Hao, Conghui Wang, Xiaocui Zhang, Zhen Li, Hongfang Shao, Hongxiang Zeng, Zhuo Yu, Li Xie, Fangzhen Xia, Feifei Zhang, Xiaoye Liu, Yaping Zhang, Haishan Jiang, Jun Zhu, Jiangbo Wan, Chun Wang, Wei Weng, Jingjing Xie, Minfang Tao, Cheng Cheng Zhang, Junling Liu, Guo-Qiang Chen, Junke Zheng
Imatinib-insensitive leukemia stem cells (LSCs) are believed to be responsible for resistance to BCR-ABL tyrosine kinase inhibitors and relapse of chronic myelogenous leukemia (CML). Identifying therapeutic targets to eradicate CML LSCs may be a strategy to cure CML. In the present study, we discovered a positive feedback loop between BCR-ABL and protein arginine methyltransferase 5 (PRMT5) in CML cells. Overexpression of
Yanli Jin, Jingfeng Zhou, Fang Xu, Bei Jin, Lijing Cui, Yun Wang, Xin Du, Juan Li, Peng Li, Ruibao Ren, Jingxuan Pan
Upfront resistance to chemotherapy and relapse following remission are critical problems in leukemia that are generally attributed to subpopulations of chemoresistant tumor cells. There are, however, limited means for prospectively identifying these subpopulations, which hinders an understanding of therapeutic resistance. BH3 profiling is a functional single-cell analysis using synthetic BCL-2 BH3 domain–like peptides that measures mitochondrial apoptotic sensitivity or “priming.” Here, we observed that the extent of apoptotic priming is heterogeneous within multiple cancer cell lines and is not the result of experimental noise. Apoptotic priming was also heterogeneous in treatment-naive primary human acute myeloid leukemia (AML) myeloblasts, and this heterogeneity decreased in chemotherapy-treated AML patients. The priming of the most apoptosis-resistant tumor cells, rather than the median priming of the population, best predicted patient response to induction chemotherapy. For several patients, these poorly primed subpopulations of AML tumor cells were enriched for antiapoptotic proteins. Developing techniques to identify and understand these apoptosis-insensitive subpopulations of tumor cells may yield insights into clinical chemoresistance and potentially improve therapeutic outcomes in AML.
Patrick D. Bhola, Brenton G. Mar, R. Coleman Lindsley, Jeremy A. Ryan, Leah J. Hogdal, Thanh Trang Vo, Daniel J. DeAngelo, Ilene Galinsky, Benjamin L. Ebert, Anthony Letai
Reducing expression of the fetal hemoglobin (HbF) repressor BCL11A leads to a simultaneous increase in γ-globin expression and reduction in β-globin expression. Thus, there is interest in targeting BCL11A as a treatment for β-hemoglobinopathies, including sickle cell disease (SCD) and β-thalassemia. Here, we found that using optimized shRNAs embedded within an miRNA (shRNAmiR) architecture to achieve ubiquitous knockdown of BCL11A profoundly impaired long-term engraftment of both human and mouse hematopoietic stem cells (HSCs) despite a reduction in nonspecific cellular toxicities. BCL11A knockdown was associated with a substantial increase in S/G2-phase human HSCs after engraftment into immunodeficient (NSG) mice, a phenotype that is associated with HSC exhaustion. Lineage-specific, shRNAmiR-mediated suppression of BCL11A in erythroid cells led to stable long-term engraftment of gene-modified cells. Transduced primary normal or SCD human HSCs expressing the lineage-specific BCL11A shRNAmiR gave rise to erythroid cells with up to 90% reduction of BCL11A protein. These erythrocytes demonstrated 60%–70% γ-chain expression (vs. < 10% for negative control) and a corresponding increase in HbF. Transplantation of gene-modified murine HSCs from Berkeley sickle cell mice led to a substantial improvement of sickle-associated hemolytic anemia and reticulocytosis, key pathophysiological biomarkers of SCD. These data form the basis for a clinical trial application for treating sickle cell disease.
Christian Brendel, Swaroopa Guda, Raffaele Renella, Daniel E. Bauer, Matthew C. Canver, Young-Jo Kim, Matthew M. Heeney, Denise Klatt, Jonathan Fogel, Michael D. Milsom, Stuart H. Orkin, Richard I. Gregory, David A. Williams
Potent CD19-directed immunotherapies, such as chimeric antigen receptor T cells (CART) and blinatumomab, have drastically changed the outcome of patients with relapsed/refractory B cell acute lymphoblastic leukemia (B-ALL). However, CD19-negative relapses have emerged as a major problem that is observed in approximately 30% of treated patients. Developing approaches to preventing and treating antigen-loss escapes would therefore represent a vertical advance in the field. Here, we found that in primary patient samples, the IL-3 receptor α chain CD123 was highly expressed on leukemia-initiating cells and CD19-negative blasts in bulk B-ALL at baseline and at relapse after CART19 administration. Using intravital imaging in an antigen-loss CD19-negative relapse xenograft model, we determined that CART123, but not CART19, recognized leukemic blasts, established protracted synapses, and eradicated CD19-negative leukemia, leading to prolonged survival. Furthermore, combining CART19 and CART123 prevented antigen-loss relapses in xenograft models. Finally, we devised a dual CAR-expressing construct that combined CD19- and CD123-mediated T cell activation and demonstrated that it provides superior in vivo activity against B-ALL compared with single-expressing CART or pooled combination CART. In conclusion, these findings indicate that targeting CD19 and CD123 on leukemic blasts represents an effective strategy for treating and preventing antigen-loss relapses occurring after CD19-directed therapies
Marco Ruella, David M. Barrett, Saad S. Kenderian, Olga Shestova, Ted J. Hofmann, Jessica Perazzelli, Michael Klichinsky, Vania Aikawa, Farzana Nazimuddin, Miroslaw Kozlowski, John Scholler, Simon F. Lacey, Jan J. Melenhorst, Jennifer J.D. Morrissette, David A. Christian, Christopher A. Hunter, Michael Kalos, David L. Porter, Carl H. June, Stephan A. Grupp, Saar Gill
Pain is a life-long symptom in sickle cell disease (SCD) and a predictor of disease progression and mortality, but little is known about its molecular mechanisms. Here, we characterized pain in a targeted knockin mouse model of SCD (TOW mouse) that exclusively expresses human alleles encoding normal α- and sickle β-globin. TOW mice exhibited ongoing spontaneous pain behavior and increased sensitivity to evoked pain compared with littermate control mice expressing normal human hemoglobins. PKCδ activation was elevated in the superficial laminae of the spinal cord dorsal horn in TOW mice, specifically in GABAergic inhibitory neurons. Functional inhibition and neuron-specific silencing of PKCδ attenuated spontaneous pain, mechanical allodynia, and heat hyperalgesia in TOW mice. Furthermore, we took a hematopoietic stem cell transplantation approach to generating a SCD model in PKCδ-deficient mice. Neither spontaneous pain nor evoked pain was detected in the mice lacking PKCδ despite full establishment of SCD phenotypes. These findings support a critical role of spinal PKCδ in the development of chronic pain in SCD, which may become a potential target for pharmacological interventions.
Ying He, Diana J. Wilkie, Jonathan Nazari, Rui Wang, Robert O. Messing, Joseph DeSimone, Robert E. Molokie, Zaijie Jim Wang
Hyperactivation of the mTOR pathway impairs hematopoietic stem cell (HSC) functions and promotes leukemogenesis. mTORC1 and mTORC2 differentially control normal and leukemic stem cell functions. mTORC1 regulates p70 ribosomal protein S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E–binding (eIF4E-binding) protein 1 (4E-BP1), and mTORC2 modulates AKT activation. Given the extensive crosstalk that occurs between mTORC1 and mTORC2 signaling pathways, we assessed the role of the mTORC1 substrate S6K1 in the regulation of both normal HSC functions and in leukemogenesis driven by the mixed lineage leukemia (MLL) fusion oncogene MLL-AF9. We demonstrated that S6K1 deficiency impairs self-renewal of murine HSCs by reducing p21 expression. Loss of S6K1 also improved survival in mice transplanted with MLL-AF9–positive leukemic stem cells by modulating AKT and 4E-BP1 phosphorylation. Taken together, these results suggest that S6K1 acts through multiple targets of the mTOR pathway to promote self-renewal and leukemia progression. Given the recent interest in S6K1 as a potential therapeutic target in cancer, our results further support targeting this molecule as a potential strategy for treatment of myeloid malignancies.
Joydeep Ghosh, Michihiro Kobayashi, Baskar Ramdas, Anindya Chatterjee, Peilin Ma, Raghuveer Singh Mali, Nadia Carlesso, Yan Liu, David R. Plas, Rebecca J. Chan, Reuben Kapur
BACKGROUND. Clinical laboratory tests are now being prescribed and made directly available to consumers through retail outlets in the USA. Concerns with these test have been raised regarding the uncertainty of testing methods used in these venues and a lack of open, scientific validation of the technical accuracy and clinical equivalency of results obtained through these services.
METHODS. We conducted a cohort study of 60 healthy adults to compare the uncertainty and accuracy in 22 common clinical lab tests between one company offering blood tests obtained from finger prick (Theranos) and 2 major clinical testing services that require standard venipuncture draws (Quest and LabCorp). Samples were collected in Phoenix, Arizona, at an ambulatory clinic and at retail outlets with point-of-care services.
RESULTS. Theranos flagged tests outside their normal range 1.6× more often than other testing services (
CONCLUSION. While laboratory practice standards exist to control this variability, the disparities between testing services we observed could potentially alter clinical interpretation and health care utilization. Greater transparency and evaluation of testing technologies would increase their utility in personalized health management.
FUNDING. This work was supported by the Icahn Institute for Genomics and Multiscale Biology, a gift from the Harris Family Charitable Foundation (to J.T. Dudley), and grants from the NIH (R01 DK098242 and U54 CA189201, to J.T. Dudley, and R01 AG046170 and U01 AI111598, to E.E. Schadt).
Brian A. Kidd, Gabriel Hoffman, Noah Zimmerman, Li Li, Joseph W. Morgan, Patricia K. Glowe, Gregory J. Botwin, Samir Parekh, Nikolina Babic, Matthew W. Doust, Gregory B. Stock, Eric E. Schadt, Joel T. Dudley
Posttranscriptional control of gene expression is important for defining both normal and pathological cellular phenotypes. In vitro, RNA-binding proteins (RBPs) have recently been shown to play important roles in posttranscriptional regulation; however, the contribution of RBPs to cell specification is not well understood. Here, we determined that the RBP insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) is specifically overexpressed in mixed lineage leukemia–rearranged (MLL-rearranged) B-acute lymphoblastic leukemia (B-ALL), which constitutes a subtype of this malignancy associated with poor prognosis and high risk of relapse. IGF2BP3 was required for the survival of B-ALL cell lines, as knockdown led to decreased proliferation and increased apoptosis. Enforced expression of IGF2BP3 provided murine BM cells with a strong survival advantage, led to proliferation of hematopoietic stem and progenitor cells, and skewed hematopoietic development to the B cell/myeloid lineage. Cross-link immunoprecipitation and high throughput sequencing uncovered the IGF2BP3-regulated transcriptome, which includes oncogenes
Jayanth Kumar Palanichamy, Tiffany M. Tran, Jonathan M. Howard, Jorge R. Contreras, Thilini R. Fernando, Timothy Sterne-Weiler, Sol Katzman, Masoud Toloue, Weihong Yan, Giuseppe Basso, Martina Pigazzi, Jeremy R. Sanford, Dinesh S. Rao