Ongoing Dll4-Notch signaling is required for T-cell homeostasis in the adult thymus.

The essential role of the Delta-like ligand 4 (Dll4)-Notch signaling pathway in T-lymphocyte development is well established. It has been shown that specific inactivation of Dll4 on thymic stromal cells during early post-natal development leads to a deregulation in T-cell differentiation. However, whether ongoing Dll4-Notch signaling is required for T-cell development in the adult thymus is unknown. The use of anti-Dll4 Abs allowed us to confirm and expand previous studies by examining the kinetics and the reversibility of Dll4-Notch signaling blockade in T-cell development in adult mice. We found that anti-Dll4 treatment reduced thymic cellularity after 7 days, as a consequence of a developmental delay in T-cell maturation at the pro-T-cell double negative 1 (CD4(-) CD8(-) c-kit(+) CD44(+) CD25(-) ) stage, leading to decreased numbers of immature double-positive (CD4(+) CD8(+) ) T cells without affecting the frequency of mature single positive CD4(+) and CD8(+) thymocytes, while promoting alternative thymic B-cell expansion. This cellular phenotype was similarly observed in both young adult and aged mice (>1.5 years), extending our understanding of the ongoing role for Dll4-Notch signaling during T-cell development in the adult thymus. Finally, after cessation of Dll4 Ab treatment, thymic cellularity and thymocyte subset ratios returned to normal levels, indicating reversibility of this phenotype in both adult and aged mice, which has important implications for potential clinical use of Dll4-Notch inhibitors.

Blockade of Dll4 inhibits tumour growth by promoting non-productive angiogenesis.

Tumour growth requires accompanying expansion of the host vasculature, with tumour progression often correlated with vascular density. Vascular endothelial growth factor (VEGF) is the best-characterized inducer of tumour angiogenesis. We report that VEGF dynamically regulates tumour endothelial expression of Delta-like ligand 4 (Dll4), which was previously shown to be absolutely required for normal embryonic vascular development. To define Dll4 function in tumour angiogenesis, we manipulated this pathway in murine tumour models using several approaches. Here we show that blockade resulted in markedly increased tumour vascularity, associated with enhanced angiogenic sprouting and branching. Paradoxically, this increased vascularity was non-productive-as shown by poor perfusion and increased hypoxia, and most importantly, by decreased tumour growth-even for tumours resistant to anti-VEGF therapy. Thus, VEGF-induced Dll4 acts as a negative regulator of tumour angiogenesis; its blockade results in a striking uncoupling of tumour growth from vessel density, presenting a novel therapeutic approach even for tumours resistant to anti-VEGF therapies.

CD22-targeted CD28 bispecific antibody enhances antitumor efficacy of odronextamab in refractory diffuse large B cell lymphoma models.

Although many patients with diffuse large B cell lymphoma (DLBCL) may achieve a complete response to frontline chemoimmunotherapy, patients with relapsed/refractory disease typically have poor outcomes. Odronextamab, a CD20xCD3 bispecific antibody that provides "signal 1" through the activation of the T cell receptor/CD3 complex, has exhibited early, promising activity for patients with highly refractory DLBCL in phase 1 trials. However, not all patients achieve complete responses, and many relapse, thus representing a high unmet medical need. Here, we investigated whether adding a costimulatory "signal 2" by engaging CD28 receptors on T cells could augment odronextamab activity. We demonstrate that REGN5837, a bispecific antibody that cross-links CD22-expressing tumor cells with CD28-expressing T cells, enhances odronextamab by potentiating T cell activation and cytolytic function. In preclinical DLBCL studies using human immune system-reconstituted animals, REGN5837 promotes the antitumor activity of odronextamab and induces intratumoral expansion of reprogrammable T cells while skewing away from a dysfunctional state. Although REGN5837 monotherapy shows limited activity and no toxicity in primate studies, it augments T cell activation when dosed in combination with odronextamab. In addition, analysis of non-Hodgkin lymphoma clinical samples reveals an increase in CD28+CD8+ T cells after odronextamab treatment, demonstrating the presence of a population that could potentially be targeted by REGN5837. Collectively, our data demonstrate that REGN5837 can markedly enhance the antitumor activity of odronextamab in preclinical NHL models, and the combination of these two bispecific antibodies may provide a chemotherapy-free approach for the treatment of DLBCL.

TGF-{beta} maintains dormancy of prostatic stem cells in the proximal region of ducts.

We have previously shown that prostatic stem cells are located in the proximal region of mouse prostatic ducts. Here, we show that this region responds differently to transforming growth factor (TGF)-beta than the distal ductal region and that under physiological conditions androgens and TGF-beta are crucial overall regulators of prostatic tissue homeostasis. This conclusion is supported by the observations showing that high levels of TGF-beta signaling are present in the quiescent proximal region of ducts in an androgen-replete animal and that cells in this region overexpress Bcl-2, which protects them from apoptosis. Moreover, androgen ablation reverses the proximal-distal TGF-beta signaling gradient, leading to an increase in TGF-beta signaling in the unprotected distal region (low Bcl-2 expression). This reversal of TGF-beta-mediated signaling accompanies apoptosis of cells in the distal region and gland involution after androgen withdrawal. A physiological TGF-beta signaling gradient (high proximally and low distally) and its functional correlates are restored after androgen replenishment. In addition to highlighting the regulatory role of androgens and TGF-beta, these findings may have important implications for the deregulation of the stem cell compartment in the etiology of proliferative prostatic diseases.

Proximal location of mouse prostate epithelial stem cells: a model of prostatic homeostasis.

Stem cells are believed to regulate normal prostatic homeostasis and to play a role in the etiology of prostate cancer and benign prostatic hyperplasia. We show here that the proximal region of mouse prostatic ducts is enriched in a subpopulation of epithelial cells that exhibit three important attributes of epithelial stem cells: they are slow cycling, possess a high in vitro proliferative potential, and can reconstitute highly branched glandular ductal structures in collagen gels. We propose a model of prostatic homeostasis in which mouse prostatic epithelial stem cells are concentrated in the proximal region of prostatic ducts while the transit-amplifying cells occupy the distal region of the ducts. This model can account for many biological differences between cells of the proximal and distal regions, and has implications for prostatic disease formation.

Blockade of Dll4 inhibits tumour growth by promoting non-productive angiogenesis.

Tumour growth requires accompanying expansion of the host vasculature, with tumour progression often correlated with vascular density. Vascular endothelial growth factor (VEGF) is the best-characterized inducer of tumour angiogenesis. We report that VEGF dynamically regulates tumour endothelial expression of Delta-like ligand 4 (Dll4), which was previously shown to be absolutely required for normal embryonic vascular development. To define Dll4 function in tumour angiogenesis, we manipulated this pathway in murine tumour models using several approaches. Here we show that blockade resulted in markedly increased tumour vascularity, associated with enhanced angiogenic sprouting and branching. Paradoxically, this increased vascularity was nonproductive--as shown by poor perfusion and increased hypoxia, and most importantly, by decreased tumour growth--even for tumours resistant to anti-VEGF therapy. Thus, VEGF-induced Dll4 acts as a negative regulator of tumour angiogenesis; its blockade results in a striking uncoupling of tumour growth from vessel density, presenting a novel therapeutic approach even for tumours resistant to anti-VEGF therapies.

Preclinical Activity of the Novel Anti-Prolactin Receptor (PRLR) Antibody-Drug Conjugate REGN2878-DM1 in PRLR-Positive Breast Cancers.

The Prolactin Receptor (PRLR) is a type 1 cytokine receptor that is expressed in a subset of breast cancers and may contribute to its pathogenesis. It is relatively overexpressed in approximately 25% of human breast tumors while expressed at low levels in some normal human tissues including the mammary gland. We developed an anti-PRLR antibody-drug conjugate (ADC), to target PRLR-positive breast cancer. REGN2878-DM1 is comprised of a fully human high-affinity function-blocking anti-PRLR IgG1 antibody (REGN2878) conjugated via a noncleavable SMCC linker to the cytotoxic maytansine derivative DM1. Both unconjugated REGN2878 and conjugated REGN2878-DM1 block PRL-mediated activation in vitro and are rapidly internalized into lysosomes. REGN2878-DM1 induces potent cell-cycle arrest and cytotoxicity in PRLR-expressing tumor cell lines. In vivo, REGN2878-DM1 demonstrated significant antigen-specific antitumor activity against breast cancer xenograft models. In addition, REGN2878-DM1 showed additive activity when combined with the antiestrogen agent fulvestrant. These results illustrate promising antitumor activity against PRLR-positive breast cancer xenografts and support the evaluation of anti-PRLR ADCs as potential therapeutic agents in breast cancer. Mol Cancer Ther; 16(7); 1299-311. ©2017 AACR.

Dll4 Blockade in Stromal Cells Mediates Antitumor Effects in Preclinical Models of Ovarian Cancer.

The Notch ligand delta-like 4 (Dll4) has been identified as a promising target in tumor angiogenesis in preclinical studies, and Dll4 inhibitors have recently entered clinical trials for solid tumors, including ovarian cancers. In this study, we report the development of REGN421 (enoticumab), a fully human IgG1 monoclonal antibody that binds human Dll4 with sub-nanomolar affinity and inhibits Notch signaling. Administering REGN421 to immunodeficient mice engineered to express human Dll4 inhibited the growth of several human tumor xenografts in association with the formation of nonfunctional tumor blood vessels. In ovarian tumor xenograft models, Dll4 was expressed specifically by the tumor endothelium, and Dll4 blockade by human-specific or mouse-specific Dll4 antibodies exerted potent antitumor activity, which relied entirely on targeting Dll4 expressed by tumor stromal cells but not by the tumor cells themselves. However, Dll4 blockade reduced Notch signaling in both blood vessels and tumor cells surrounding the blood vessels, suggesting that endothelial-expressed Dll4 might induce Notch signaling in adjacent ovarian tumor cells. The antitumor effects of targeting Dll4 were augmented significantly by simultaneous inhibition of VEGF signaling, whereas this combined blockade reversed normal organ vascular changes induced by Dll4 blockade alone. Overall, our findings deepen the rationale for antibody-based strategies to target Dll4 in ovarian cancers, especially in combination with VEGF blockade.

Proximal prostatic stem cells are programmed to regenerate a proximal-distal ductal axis.

Prostate carcinoma and benign prostatic hypertrophy may both originate in stem cells, highlighting the importance of the characterization of these cells. The prostate gland contains a network of ducts each of which consists of a proximal (adjacent to the urethra), an intermediate, and a distal region. Here, we report that two populations of cells capable of regenerating prostatic tissue in an in vivo prostate reconstitution assay are present in different regions of prostatic ducts. The first population (with considerable growth potential) resides in the proximal region of ducts and in the urethra, and the survival of these cells does not require the presence of androgens. The second population (with more limited growth potential) is found in the remaining ductal regions and requires androgen for survival. In addition, we find that primitive proximal prostate cells that are able to regenerate functional prostatic tissue in vivo are also programmed to re-establish a proximal-distal ductal axis. Similar to their localization in the intact prostate, cells with the highest regenerative capacity are found in the proximal region of prostatic ducts formed in an in vivo prostate reconstitution assay. The primitive proximal cells can be passaged through four generations of subrenal capsule grafts. Together, these novel findings illustrate features of primitive prostate cells that may have implications for the development of therapies for treating proliferative prostatic diseases.

Sca-1 expression identifies stem cells in the proximal region of prostatic ducts with high capacity to reconstitute prostatic tissue.

We previously showed that prostatic stem cells are concentrated in the proximal regions of prostatic ducts. We now report that these stem cells can be purified from isolated proximal duct regions by virtue of their high expression of the cell surface protein stem cell antigen 1 (Sca-1). In an in vivo prostate reconstitution assay, the purified Sca-1-expressing cell population isolated from the proximal region of ducts was more effective in generating prostatic tissue than a comparable population of Sca-1-depleted cells (203.0 +/- 83.1 mg vs. 11.9 +/- 9.2 mg) or a population of Sca-1-expressing cells isolated from the remaining regions of ducts (transit-amplifying cells) (31.9 +/- 24.1 mg). Almost all of the proliferative capacity of the proximal duct Sca-1-expressing cell population resides within the fraction of cells that express high levels of Sca-1 (top one-third), with the proximal region of prostatic ducts containing 7.2-fold more Sca-1(high) cells than the remaining regions. More than 60% of the high-expressing cells coexpress alpha6 integrin and the anti-apoptotic factor Bcl-2, markers that are also characteristic of stem cells of other origins. Further stratification of the phenotype of the stem cells may enable the development of rational therapies for treating prostate cancer and benign prostatic hyperplasia.

Differentiation and stromal-induced growth promotion of murine prostatic tumors.

BACKGROUND: We have derived a panel of p53-null prostatic "basal" and "luminal" epithelial cell lines and their ras transformed counterparts to study stromal/epithelial interactions and the properties of tumors arising from "basal" and "luminal" cells. METHODS: Previously derived normal murine prostatic "basal" epithelial (PE-B-1) and "luminal" epithelial (PE-L-1) cell lines were transformed with N-Ras. These lines and a spontaneously transformed "luminal" cell line were inoculated subcutaneously or orthotopically into athymic mice, alone or in combination with normal prostatic smooth muscle cells (SMC). RESULTS: All transformed lines formed subcutaneous tumors. SMC significantly enhanced the growth rate of the tumors arising from the "basal" and one of the "luminal" cell lines. The transformed "basal" line gave rise to tumors expressing both "basal" and "luminal" cytokeratins. CONCLUSIONS: Prostatic SMC promote the growth of transformed epithelial cells, suggesting that prostatic stroma may promote tumor development. Furthermore, transformed "basal" cells give rise to tumors containing "luminal" cells, suggesting that although most human tumors have a "luminal" phenotype, they may originate from transformed "basal" cells.

Basic fibroblast growth factor modulates the expression of glycophorin A and c-kit and inhibits erythroid differentiation in K562 cells.

Basic fibroblast growth factor (bFGF) is produced by bone marrow stromal cells as well as by normal and leukemic hematopoietic cells. In this study, we examine the direct effects of bFGF on erythroid differentiation in K562 cells in order to determine whether bFGF can promote the expression of a primitive phenotype. Low levels of bFGF inhibited erythroid differentiation as evidenced by decreased expression of glycophorin A and increased expression of c-kit. bFGF also increased both the numbers and the sizes of colonies of K562 cells in soft agar assays. The addition of TGF-beta to these cells induced erythroid differentiation which resulted in an increase in glycophorin A and a decrease in c-kit. The simultaneous addition of bFGF and TGF-beta to K562 cells prevented both the TGF-beta-mediated increase in glycophorin A expression and the decrease in c-kit expression associated with erythroid differentiation. bFGF antagonised the TGF-beta-mediated promotion of erythroid differentiation in K562 cells in a dose dependent manner and these two cytokines counteracted each other on an approximately molar basis. These results indicate that bFGF alone increases expression of c-kit and promotes a primitive phenotype in K562 cells. In addition, bFGF counteracts the effects of differentiation-inducing cytokines, such as TGF-beta, on hematopoietic cells. It is therefore possible that enhanced production of bFGF by leukemic cells could contribute to their neoplastic phenotype by opposing the effects of negative regulators or cytokines that induce differentiation.

Stromal/epithelial interactions of murine prostatic cell lines in vivo: a model for benign prostatic hyperplasia and the effect of doxazosin on tissue size.

BACKGROUND: One of the major constraints in elucidating the mechanisms involved in the etiology of benign prostatic hyperplasia (BPH) is the lack of suitable model systems that are readily manipulable in vitro and in vivo. To address this issue, we have used murine prostatic cell lines to establish a novel in vivo model for studying prostatic cell interactions. METHODS: Luminal, basal, and smooth muscle (SM) cell lines were inoculated alone or in combinations under the renal capsule of intact or castrated male mice, and the growth and composition of prostatic tissue in the absence or presence of doxazosin was determined. RESULTS: Both the luminal and basal cell lines reconstituted prostatic tissue if co-inoculated under the renal capsule with normal SM cells, whereas none of the lines formed significant tissue when inoculated alone. Luminal cells produced and secreted prostatic secretory products. The growth of prostatic tissue formed from co-inoculation of basal and SM cells was androgen responsive. In addition, a significant reduction in prostatic tissue was noted in animals treated with doxazosin. CONCLUSION: We have established an in vivo model that uses prostatic epithelial and SM cell lines for investigating cellular interactions between epithelial and SM cells that regulate prostatic growth and function. This model will be useful for delineating the mechanisms by which prostatic cells interact and in determining the efficacy of new approaches aimed at interfering with prostatic stromal/epithelial interactions that result in abnormal cellular proliferation.

Fibroblast growth factor receptor-1 is expressed by endothelial progenitor cells.

Recent experiments show that hematopoietic progenitor cell populations contain endothelial precursor cells. We have isolated a population of CD34(+) cells that expresses fibroblast growth factor receptor-1 (FGFR-1) and that differentiates into endothelial cells in vitro. We find that 4.5% +/- 2.1% of CD34(+) cells isolated from bone marrow, cord blood, and mobilized peripheral blood express FGFR-1 and that viable CD34(+)FGFR(+) cells are small, with little granularity, and express both primitive hematopoietic and endothelial markers on their surface. The primitive hematopoietic markers AC133, c-kit, and Thy-1 are coexpressed by 75%, 85%, and 64% of CD34(+)FGFR(+) cells, respectively. Most of the CD34(+)FGFR(+) cells also express antigens found on endothelial cells, such as CD31, vascular endothelial growth factor receptor-2, and the endothelial-specific cell surface marker, vascular endothelial cadherin (VE-cadherin), whereas 56% to 60% of the cells express Tie, Tek, and the endothelial-specific marker, P1H12. The CD34(+)FGFR(+) population is enriched in cells expressing endothelial-specific antigens compared with the CD34(+) population. Isolated CD34(+)FGFR(+) cells grow slowly in culture, are stimulated by fibroblast growth factor-2 and vascular endothelial growth factor, and give rise to cells that express von Willebrand factor and VE-cadherin and that incorporate acetylated low-density lipoprotein. These experiments show that FGFR-1 is expressed by a subpopulation of CD34(+) cells that give rise to endothelial cells in vitro, indicating that this population contains endothelial stem/progenitor cells.