Gerald Wertheim

A novel doxycycline-inducible system for the transgenic analysis of mammary gland biology

Normal developmental events such as puberty, pregnancy, and parity influence the susceptibility of the mammary gland to tumorigenesis in both humans and rodent model systems. Unfortunately, constitutive transgenic mouse models that rely on mammary‐specific promoters to control transgene expression have limited utility for studying the effect of developmental events on breast cancer risk since the hormonal signals governing these events also markedly influence transgene expression levels. A novel transgenic mouse system is described that uses the MMTV‐LTR to drive expression of the reverse tetracycline‐dependent transactivator rtTA. Transgenic mice expressing rtTA in the mammary epithelium were crossed with reporter lines bearing tet operator‐controlled transgenes. We tested the ability to spatially, temporally, and quantitatively control reporter gene expression after administration of doxycycline to bitransgenic mice. Transgene expression using this system can be rapidly induced and deinduced, is highly mammary specific, can be reproducibly titrated over a wide range of expression levels, and is essentially undetectable in the uninduced state. Homogeneous transgene expression throughout the mammary epithelium can be achieved. This system permits transgene expression to be restricted to any desired stage of postnatal mammary gland development. We have developed a mammary‐specific, doxycycline‐inducible transgenic mouse model for studying the effect of mammary gland development on transgene‐mediated phenotypes. Unlike other mammary‐specific, transgenic systems that have been described, this system combines spatially homogeneous transgene expression in the mammary epithelium during puberty, pregnancy, lactation, and involution with the use of an orally administered, inexpensive, and widely available inducing agent. This system offers new opportunities for the transgenic analysis of mammary gland biology in vivo.—Gunther, E. J., Belka, G. K., Wertheim, G. B. W., Wang, J., Hartman, J. L., Boxer, R. B., Chodosh, L. A. A novel doxycycline‐inducible system for the transgenic analysis of mammary gland biology. FASEB J. 16, 283–292 (2002)

Identification of Predictive Biomarkers for Cytokine Release Syndrome after Chimeric Antigen Receptor T-cell Therapy for Acute Lymphoblastic Leukemia.

UNLABELLED Chimeric antigen receptor (CAR)-modified T cells with anti-CD19 specificity are a highly effective novel immune therapy for relapsed/refractory acute lymphoblastic leukemia. Cytokine release syndrome (CRS) is the most significant and life-threatening toxicity. To improve understanding of CRS, we measured cytokines and clinical biomarkers in 51 CTL019-treated patients. Peak levels of 24 cytokines, including IFNγ, IL6, sgp130, and sIL6R, in the first month after infusion were highly associated with severe CRS. Using regression modeling, we could accurately predict which patients would develop severe CRS with a signature composed of three cytokines. Results were validated in an independent cohort. Changes in serum biochemical markers, including C-reactive protein and ferritin, were associated with CRS but failed to predict development of severe CRS. These comprehensive profiling data provide novel insights into CRS biology and, importantly, represent the first data that can accurately predict which patients have a high probability of becoming critically ill. SIGNIFICANCE CRS is the most common severe toxicity seen after CAR T-cell treatment. We developed models that can accurately predict which patients are likely to develop severe CRS before they become critically ill, which improves understanding of CRS biology and may guide future cytokine-directed therapy. Cancer Discov; 6(6); 664-79. ©2016 AACR.See related commentary by Rouce and Heslop, p. 579This article is highlighted in the In This Issue feature, p. 561.

Persistence of long-lived plasma cells and humoral immunity in individuals responding to CD19-directed CAR T-cell therapy.

The mechanisms underlying the maintenance of long-lasting humoral immunity are not well understood. Studies in mice indicate that plasma cells (PCs) can survive up to a lifetime, even in the absence of regeneration by B cells, implying the presence of long-lived PCs as a mechanism for long-lasting immunity. Evidence from humans treated with anti-CD20, which depletes circulating B cells, also suggests B-cell-independent long-term survival of some PCs. On the other hand, antibody responses may be sustained solely by short-lived PCs with repopulation from clonally related memory B cells. To explore PC longevity and humoral immunity in humans, we investigated the fate of PCs and their antibodies in adult and pediatric patients who received chimeric antigen receptor-based adoptive T-cell immunotherapy targeting CD19 to treat B-cell lineage malignancies (CTL019). Treatment with CTL019 is frequently associated with B-cell aplasia that can persist for years. Serum antibody titers to vaccine-related antigens were measured, and quantitative assessment of B cells and PCs in blood and bone marrow was performed at various time points before and after CTL019 therapy. While total serum immunoglobulin concentrations decline following CTL019-induced B-cell aplasia, several vaccine/pathogen-specific serum immunoglobulin G and A (IgG and IgA) titers remain relatively stable for at least 6 and 12 months posttreatment, respectively. Analysis of bone marrow biopsies after CTL019 revealed 8 patients with persistence of antibody-secreting PCs at least 25 months post-CTL019 infusion despite absence of CD19(+)CD20(+) B cells. These results provide strong evidence for the existence of memory B-cell-independent, long-lived PCs in humans that contribute to long-lasting humoral immunity.

The Notch1 transcriptional activation domain is required for development and reveals a novel role for Notch1 signaling in fetal hematopoietic stem cells

Notch1 is required to generate the earliest embryonic hematopoietic stem cells (HSCs); however since Notch-deficient embryos die early in gestation, additional functions for Notch in embryonic HSC biology have not been described. We used two complementary genetic models to address this important biological question. Unlike Notch1-deficient mice, mice lacking the conserved Notch1 transcriptional activation domain (TAD) show attenuated Notch1 function in vivo and survive until late gestation, succumbing to multiple cardiac abnormalities. Notch1 TAD-deficient HSCs emerge and successfully migrate to the fetal liver but are decreased in frequency by embryonic day 14.5. In addition, TAD-deficient fetal liver HSCs fail to compete with wild-type HSCs in bone marrow transplant experiments. This phenotype is independently recapitulated by conditional knockout of Rbpj, a core Notch pathway component. In vitro analysis of Notch1 TAD-deficient cells shows that the Notch1 TAD is important to properly assemble the Notch1/Rbpj/Maml trimolecular transcription complex. Together, these studies reveal an essential role for the Notch1 TAD in fetal development and identify important cell-autonomous functions for Notch1 signaling in fetal HSC homeostasis.

The Snf1-related kinase, Hunk, is essential for mammary tumor metastasis

We previously identified a SNF1/AMPK-related protein kinase, Hunk, from a mammary tumor arising in an MMTV-neu transgenic mouse. The function of this kinase is unknown. Using targeted deletion in mice, we now demonstrate that Hunk is required for the metastasis of c-myc-induced mammary tumors, but is dispensable for normal development. Reconstitution experiments revealed that Hunk is sufficient to restore the metastatic potential of Hunk-deficient tumor cells, as well as defects in migration and invasion, and does so in a manner that requires its kinase activity. Consistent with a role for this kinase in the progression of human cancers, the human homologue of Hunk is overexpressed in aggressive subsets of carcinomas of the ovary, colon, and breast. In addition, a murine gene expression signature that distinguishes Hunk-wild type from Hunk-deficient mammary tumors predicts clinical outcome in women with breast cancer in a manner consistent with the pro-metastatic function of Hunk in mice. These findings identify a direct role for Hunk kinase activity in metastasis and establish an in vivo function for this kinase.

Identification of Flt3⁺CD150⁻ myeloid progenitors in adult mouse bone marrow that harbor T lymphoid developmental potential.

Common myeloid progenitors (CMPs) were first identified as progenitors that were restricted to myeloid and erythroid lineages. However, it was recently demonstrated that expression of both lymphoid- and myeloid-related genes could be detected in myeloid progenitors. Furthermore, these progenitors were able to give rise to T and B lymphocytes, in addition to myeloid cells. Yet, it was not known whether these progenitors were multipotent at the clonogenic level or there existed heterogeneity within these progenitors with different lineage potential. Here we report that previously defined CMPs possess T-lineage potential, and that this is exclusively found in the Flt3(+)CD150(-) subset of CMPs at the clonal level. In contrast, we did not detect B-lineage potential in CMP subsets. Therefore, these Flt3(+)CD150(-) myeloid progenitors were T/myeloid potent. Yet, Flt3(+)CD150(-) myeloid progenitors are not likely to efficiently traffic to the thymus and contribute to thymopoiesis under normal conditions because of the lack of CCR7 and CCR9 expression. Interestingly, both Flt3(+)CD150(-) and Flt3(-)CD150(-) myeloid progenitors are susceptible to Notch1-mediated T-cell acute lymphoblastic leukemia (T-ALL). Hence, gain-of-function Notch1 mutations occurring in developing myeloid progenitors, in addition to known T-lineage progenitors, could lead to T-ALL oncogenesis.

Nucleophosmin (NPM1) mutations in acute myeloid leukemia: an ongoing (cytoplasmic) tale of dueling mutations and duality of molecular genetic testing methodologies.

This Commentary highlights two articles that focus on molecular techniques to identify mutations in nucleophosmin (NPM1), which is the most frequently mutated gene in cytogenetically normal acute myeloid leukemia (CN-AML)

Repeated loss of target surface antigen after immunotherapy in primary mediastinal large B cell lymphoma.

Washington, District of Columbia; Hematology Oncology, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts; Medicine, Pathology and Laboratory Medicine, Boston University School of Medicine, Boston, Massachusetts; Phoenicia Biosciences, Weston, Massachusetts Conflict of interest: S. Perrine: Inventor on patents related to this work. Contract grant sponsor: NIH; Contract grant numbers: 1P50 HL-118006, R01 DK-52962, R41 HL-108516, R42 HL-110727. *Correspondence to: Susan Perrine, MD, Hemoglobinopathy Thalassemia Research Unit, Boston University School of Medicine, 72 East Concord Street, L909, Boston, MA 02118. E-mail: sperrine@bu.edu Received for publication: 27 September 2016; Revised: 13 October 2016; Accepted: 17 October 2016 Published online: 20 October 2016 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ajh.24590

Ikaros Imposes a Barrier to CD8 + T Cell Differentiation by Restricting Autocrine IL-2 Production

Naive CD4+ T cells require signals from the TCR and CD28 to produce IL-2, expand, and differentiate. However, these same signals are not sufficient to induce autocrine IL-2 production by naive CD8+ T cells, which require cytokines provided by other cell types to drive their differentiation. The basis for failed autocrine IL-2 production by activated CD8+ cells is unclear. We find that Ikaros, a transcriptional repressor that silences IL-2 in anergic CD4+ T cells, also restricts autocrine IL-2 production by CD8+ T cells. We find that CD8+ T cell activation in vitro in the absence of exogenous cytokines and CD4 help leads to marked induction of Ikaros, a known repressor of the Il2 gene. Naive murine CD8 T cells haplo-insufficient for Ikzf1 failed to upregulate Ikaros, produced autocrine IL-2, and differentiated in an IL-2–dependent manner into IFN-γ–producing CTLs in response to TCR/CD28 stimulation alone. Furthermore, Ikzf1 haplo-insufficient CD8+ T cells were more effective at controlling Listeria infection and B16 melanoma growth in vivo, and they could provide help to neighboring, non-IL-2–producing cells to differentiate into IFN-γ–producing effectors. Therefore, by repressing autocrine IL-2 production, Ikaros ensures that naive CD8+ T cells remain dependent on licensing by APCs and CD4+ T cells, and it may therefore act as a cell-intrinsic safeguard against inappropriate CTL differentiation and immunopathology.

Minimal residual disease testing to predict relapse following transplant for AML and high-grade myelodysplastic syndromes.

Evaluation of: Lange T, Hubmann M, Burkhardt R et al. Monitoring of WT1 expression in PB and CD34+ donor chimerism of BM predicts early relapse in AML and MDS patients after hematopoietic cell transplantation with reduced-intensity conditioning. Leukemia 25, 498–505 (2011). Early detection of relapse is critical for patients who have undergone hematopoietic stem cell transplantation (HSCT) for acute myeloid leukemia (AML) or high-grade myelodysplastic syndromes (MDS), since therapy can be initiated while disease burden remains low. As these neoplasms represent a heterogeneous group of malignancies with distinct underlying mutations, no single genetic marker exists that both defines AML/MDS and can be exploited for sensitive detection of neoplastic cells prior to overt hematologic relapse. Conversely, the Wilms’ tumor gene (WT1) expression level is increased in blasts of most AML/MDS patients, and quantitative analysis of WT1 expression has been used to predict relapse following myeloablative HSCT. In this article, we review a recently published study evaluating the usefulness of multiple markers, including WT1 expression, for predicting relapse in AML/MDS patients following reduced-intensity conditioning nonmyeloablative HSCT.