HER2 and HLA-A*02 dual CAR-T cells utilize LOH in a NOT logic gate to address on-target off-tumor toxicity.

BACKGROUND: One of the major challenges in chimeric antigen receptor (CAR)-T cell therapy for solid tumors is the potential for on-target off-tumor toxicity due to the expression of CAR tumor antigens in essential tissues and organs. Here, we describe a dual CAR NOT gate incorporating an inhibitory CAR (iCAR) recognizing HLA-A*02 ("A2") that enables effective treatment with a potent HER2 activating CAR (aCAR) in the context of A2 loss of heterozygosity (LOH). METHODS: A CAR-T cell screen was conducted to identify inhibitory domains derived from natural immune receptors (iDomains) to be used in a NOT gate, to kill A2- HER2+ lung cancer cell lines but spare A2+ HER2+ lung cancer cell-lines with high specificity. The extensive analysis of lead candidates included T-cell activation and killing, assays of reversibility and durability in sequential challenges, target cell specificity in mixed 3D spheroids and 2D cultures, and the characterization of CAR expression level and cell-trafficking. RESULTS: A leukocyte immunoglobulin-like receptor B1 (LIR1) iDomain iCAR was identified as most effective in regulating the cytotoxicity of a second generation HER2 aCAR. Target transfer experiments demonstrated that the 'on' and 'off' cell state of the LIR1 NOT gate CAR-T cell is both durable and reversible. Protection required iCAR signaling and was associated with reduced aCAR and iCAR surface expression. iCAR regulation was sufficient to generate high target specificity in a 3D adjacent spheroid assay designed to model the interface between clonal A2 LOH foci and normal tissue. However, we observed significant bystander killing of A2+ cells in admix culture through aCAR dependent and independent mechanisms. LIR1 NOT gate CAR-T cells conferred protection against H1703-A2+ tumors and high efficacy against H1703-A2- tumors in-vivo. We observed that the iCAR is inactive in A2+ donors due to cis-binding, but Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) knockout of HLA-A fully restored iCAR activity. CONCLUSIONS: We have preclinically validated an iCAR NOT gate technology broadly applicable for targeting HER2 expression in the context of A2 LOH. This approach is designed to prevent off tumor toxicity while allowing highly potent antitumor activity.

Nonclinical Safety Assessment of AMG 553, an Investigational Chimeric Antigen Receptor T-Cell Therapy for the Treatment of Acute Myeloid Leukemia.

Feline McDonough Sarcoma-like tyrosine kinase 3 (FLT3), a tyrosine-protein kinase involved in hematopoiesis, is detectable on the cell surface of approximately 80% of leukemia isolates from adult patients with acute myeloid leukemia (AML). AMG 553 is an investigational chimeric antigen receptor (CAR) T-cell immunotherapy for the treatment of AML. FLT3 expression analysis and in vitro and in vivo studies were leveraged to evaluate the nonclinical safety of AMG 553. Cynomolgus monkeys administered autologous anti-FLT3 CAR T cells demonstrated no evidence of CAR T-cell-mediated toxicity, expansion, or persistence, likely due to restricted cell surface FLT3 protein expression in healthy animals. This highlights the limited value of such in vivo studies for safety assessment of the CAR T-cell modality when directed against a target with restricted expression. To complement these studies and directly evaluate the potential toxicities of eliciting T-cell-mediated cytotoxicity against cells with surface expression of FLT3 protein in vivo, data from cynomolgus monkey toxicology studies with 2 bispecific T-cell engager molecules targeting FLT3 were leveraged; findings were consistent with the targeted killing of bone marrow cells expressing cell surface FLT3. Potential AMG 553-induced cytotoxicity was assessed against a wide range of normal human primary cells and cell lines; cytotoxicity was observed against FLT3-positive AML cell lines and a percentage of primary bone marrow CD34+ cells. In conclusion, the nonclinical safety data suggest that AMG 553 can target FLT3 protein on AML cells, whereas only affecting a percentage of normal hematopoietic stem and progenitor cells, supporting clinical development.

The Genetic Landscape of Hematopoietic Stem Cell Frequency in Mice.

Prior efforts to identify regulators of hematopoietic stem cell physiology have relied mainly on candidate gene approaches with genetically modified mice. Here we used a genome-wide association study (GWAS) strategy with the hybrid mouse diversity panel to identify the genetic determinants of hematopoietic stem/progenitor cell (HSPC) frequency. Among 108 strains, we observed ∼120- to 300-fold variation in three HSPC populations. A GWAS analysis identified several loci that were significantly associated with HSPC frequency, including a locus on chromosome 5 harboring the homeodomain-only protein gene (Hopx). Hopx previously had been implicated in cardiac development but was not known to influence HSPC biology. Analysis of the HSPC pool in Hopx-/- mice demonstrated significantly reduced cell frequencies and impaired engraftment in competitive repopulation assays, thus providing functional validation of this positional candidate gene. These results demonstrate the power of GWAS in mice to identify genetic determinants of the hematopoietic system.

Prolonged fasting reduces IGF-1/PKA to promote hematopoietic-stem-cell-based regeneration and reverse immunosuppression.

Immune system defects are at the center of aging and a range of diseases. Here, we show that prolonged fasting reduces circulating IGF-1 levels and PKA activity in various cell populations, leading to signal transduction changes in long-term hematopoietic stem cells (LT-HSCs) and niche cells that promote stress resistance, self-renewal, and lineage-balanced regeneration. Multiple cycles of fasting abated the immunosuppression and mortality caused by chemotherapy and reversed age-dependent myeloid-bias in mice, in agreement with preliminary data on the protection of lymphocytes from chemotoxicity in fasting patients. The proregenerative effects of fasting on stem cells were recapitulated by deficiencies in either IGF-1 or PKA and blunted by exogenous IGF-1. These findings link the reduced levels of IGF-1 caused by fasting to PKA signaling and establish their crucial role in regulating hematopoietic stem cell protection, self-renewal, and regeneration.

Adipocytic cells augment the support of primitive hematopoietic cells in vitro but have no effect in the bone marrow niche under homeostatic conditions.

Mesenchymal stem cells (MSCs), as well as osteoblastic cells derived from these MSCs, have been shown to be key components of the hematopoietic stem cell (HSC) niche. In this study, we wished to examine whether other cell types that are known to differentiate from MSCs similarly regulate the stem cell niche, namely cells of the adipocyte lineage. Recent studies have examined the role that adipocytes play in the biology of the HSCs in different bone locations and in transplantation settings; however, none have examined their role under homeostatic conditions. We compared the ability of adipocytic and nonadipocytic cell lines to support primitive hematopoietic cells in vitro. Preadipocytic cell lines demonstrated enhanced support of hematopoietic cells. Similarly, primary bone marrow (BM) cells treated with troglitazone, a drug that enhances adipogenesis, also demonstrated augmented support over control-treated stromal cells. We further examined the effects of increased adipocyte number in vivo under homeostatic conditions using troglitazone treatment and found that these alterations had no effect on HSC frequency. Taken together, we demonstrate that cells of the adipocyte lineage promote the ability of stromal cells to support primitive hematopoietic cells in vitro, yet alterations of adipocyte number and volume in vivo have no effect. These data suggest that adipocytes are not a component of the adult BM HSC niche under homeostatic conditions.

Placental growth factor expression is required for bone marrow endothelial cell support of primitive murine hematopoietic cells.

Two distinct microenvironmental niches that regulate hematopoietic stem/progenitor cell physiology in the adult bone marrow have been proposed; the endosteal and the vascular niche. While extensive studies have been performed relating to molecular interactions in the endosteal niche, the mechanisms that regulate hematopoietic stem/progenitor cell interaction with bone marrow endothelial cells are less well defined. Here we demonstrate that endothelial cells derived from the bone marrow supported hematopoietic stem/progenitor cells to a higher degree than other endothelial or stromal cell populations. This support was dependant upon placental growth factor expression, as genetic knockdown of mRNA levels reduced the ability of endothelial cells to support hematopoietic stem/progenitor cells in vitro. Furthermore, using an in vivo model of recovery from radiation induced myelosuppression, we demonstrate that bone marrow endothelial cells were able to augment the recovery of the hematopoietic stem/progenitor cells. However, this effect was diminished when the same cells with reduced placental growth factor expression were administered, possibly owing to a reduced homing of the cells to the bone marrow vasculature. Our data suggest that placental growth factor elaborated from bone marrow endothelial cells mediates the regulatory effects of the vascular niche on hematopoietic stem/progenitor cell physiology.

Deficiency of GRP94 in the hematopoietic system alters proliferation regulators in hematopoietic stem cells.

We have previously reported that acute inducible knockout of the endoplasmic reticulum chaperone GRP94 led to an expansion of the hematopoietic stem and progenitor cell pool. Here, we investigated the effectors and mechanisms for this phenomenon. We observed an increase in AKT activation in freshly isolated GRP94-null HSC-enriched Lin(-) Sca-1(+) c-Kit(+) (LSK) cells, corresponding with higher production of PI(3,4,5)P3, indicative of PI3K activation. Treatment of GRP94-null LSK cells with the AKT inhibitor MK2206 compromised cell expansion, suggesting a causal relationship between elevated AKT activation and increased proliferation in GRP94-null HSCs. Microarray analysis demonstrated a 97% reduction in the expression of the hematopoietic cell cycle regulator Ms4a3 in the GRP94-null LSK cells, and real-time quantitative PCR confirmed this down-regulation in the LSK cells but not in the total bone marrow (BM). A further examination comparing freshly isolated BM LSK cells with spleen LSK cells, as well as BM LSK cells cultured in vitro, revealed specific down-regulation of Ms4a3 in freshly isolated BM GRP94-null LSK cells. On examining cell surface proteins that are known to regulate stem cell proliferation, we observed a reduced expression of cell surface connexin 32 (Cx32) plaques in GRP94-null LSK cells. However, suppression of Cx32 hemichannel activity in wild-type LSK cells through mimetic peptides did not lead to increased LSK cell proliferation in vitro. Two other important cell surface proteins that mediate HSC-niche interactions, specifically Tie2 and CXCR4, were not impaired by Grp94 deletion. Collectively, our study uncovers novel and unique roles of GRP94 in regulating HSC proliferation.

The endoplasmic reticulum chaperone protein GRP94 is required for maintaining hematopoietic stem cell interactions with the adult bone marrow niche.

Hematopoietic stem cell (HSC) homeostasis in the adult bone marrow (BM) is regulated by both intrinsic gene expression products and interactions with extrinsic factors in the HSC niche. GRP94, an endoplasmic reticulum chaperone, has been reported to be essential for the expression of specific integrins and to selectively regulate early T and B lymphopoiesis. In GRP94 deficient BM chimeras, multipotent hematopoietic progenitors persisted and even increased, however, the mechanism is not well understood. Here we employed a conditional knockout (KO) strategy to acutely eliminate GRP94 in the hematopoietic system. We observed an increase in HSCs and granulocyte-monocyte progenitors in the Grp94 KO BM, correlating with an increased number of colony forming units. Cell cycle analysis revealed that a loss of quiescence and an increase in proliferation led to an increase in Grp94 KO HSCs. This expansion of the HSC pool can be attributed to the impaired interaction of HSCs with the niche, evidenced by enhanced HSC mobilization and severely compromised homing and lodging ability of primitive hematopoietic cells. Transplanting wild-type (WT) hematopoietic cells into a GRP94 null microenvironment yielded a normal hematology profile and comparable numbers of HSCs as compared to WT control, suggesting that GRP94 in HSCs, but not niche cells, is required for maintaining HSC homeostasis. Investigating this, we further determined that there was a near complete loss of integrin α4 expression on the cell surface of Grp94 KO HSCs, which showed impaired binding with fibronectin, an extracellular matrix molecule known to play a role in mediating HSC-niche interactions. Furthermore, the Grp94 KO mice displayed altered myeloid and lymphoid differentiation. Collectively, our studies establish GRP94 as a novel cell intrinsic factor required to maintain the interaction of HSCs with their niche, and thus regulate their physiology.

Blocking HIF1α activity eliminates hematological cancer stem cells.

Selective targeting of cancer stem cells (CSCs) has the potential to prevent cancer relapse. Wang et al. (2011) report that hypoxia-inducible factor 1α (HIF1α) represses Notch signaling to maintain CSC subsets from lymphoma, and that blocking HIF1α activity eliminates lymphoma and human acute myeloid leukemia (AML) CSCs.

Bone marrow fat is inversely related to cortical bone in young and old subjects.

OBJECTIVE: Recent studies suggest a close local link between bone marrow adiposity and endosteal bone formation. Using magnetic resonance imaging, we examined whether the relation between the amount of marrow fat and cortical bone is present at multiple sites along the diaphyses of the long bones of young and old males and females. DESIGN: The relations between values for cortical bone area and percent marrow fat in each 5-mm section along the midthird of both femoral shafts were determined using magnetic resonance imaging in eight healthy young (aged <25 yr), and nine healthy old (aged >55 yr) men and women. RESULTS: Strong inverse correlations were observed between values for cortical bone area and percent marrow fat along the shafts of all 34 femurs; r values between -0.54 to -0.97; all P values = 0.01-0.0001. The strength of this local association was comparable in the young and the elderly and in males and females. CONCLUSION: Our results underscore the strength of the local connections between bone and marrow adiposity. Increasing our understanding of the mechanism for this association could lead to better diagnosis and treatment approaches for osteoporosis.

Pharmacologic modulation of the calcium-sensing receptor enhances hematopoietic stem cell lodgment in the adult bone marrow.

The ability of hematopoietic stem cells (HSCs) to undergo self-renewal is partly regulated by external signals originating from the stem cell niche. Our previous studies with HSCs obtained from fetal liver of mice deficient for the calcium-sensing receptor (CaR) have shown the crucial role of this receptor in HSC lodgment and engraftment in the bone marrow (BM) endosteal niche. Using a CaR agonist, Cinacalcet, we assessed the effects of stimulating the CaR on the function of murine HSCs. Our results show that CaR stimulation increases primitive hematopoietic cell activity in vitro, including growth in stromal cell cocultures, adhesion to extracellular matrix molecules such as collagen I and fibronectin, and migration toward the chemotactic stimulus, stromal cell-derived factor 1α. Receptor stimulation also led to augmented in vivo homing, CXCR4-mediated lodgment at the endosteal niche, and engraftment capabilities. These mechanisms by which stimulating the CaR dictates preferential localization of HSCs in the BM endosteal niche provide additional insights into the fundamental interrelationship between the stem cell and its niche. These studies also have implications in the area of clinical stem cell transplantation, where ex vivo modulation of the CaR may be envisioned as a strategy to enhance HSC engraftment in the BM.

Hematopoietic stem cell lodgment in the adult bone marrow stem cell niche.

Treatment of malignant blood disorders, such as leukemia, that can provide a better chance of long-term remission involves myeloablation followed by transplantation of matched donor hematopoietic stem cells (HSCs). For successful engraftment and re-establishment of hematopoiesis to occur in the recipient, the transplanted HSCs must first migrate from the blood circulation to the bone marrow (BM), a process known as homing, then localize and anchor in suitable microenvironments within the BM, a process known as lodgment. After lodgment, the specific fate of the transplanted HSCs is determined through complex, bidirectional interactions with various stromal cell components in the niche. Ultimately, these interactions dictate the clinical outcome of the transplantation. Through the use of transgenic mouse models, considerable evidence has been accumulated in an attempt to unveil the possible underlying mechanisms that govern these processes. Here, we will emphasize the major factors that are involved in the regulation of lodgment of transplanted HSCs. Specifically, we will first introduce early observations on the spatial distribution of hematopoietic progenitors within the BM, then we will discuss the soluble factors, chemokines, cell-cell interactions, and cell-matrix interactions that have been studied and known to influence the site of HSC lodgment within the BM following transplantation.

The influence of parathyroid hormone on the adult hematopoietic stem cell niche.

Adult hematopoietic stem cells (HSCs) reside in the bone marrow in stable microenvironments known as the stem cell niche. One key component of the stem cell niche is cells of the osteoblastic lineage. Factors that are known to affect osteoblast activity, such as parathyroid hormone (PTH), have also been shown to affect the HSCs. Treatment of mice with PTH has led to beneficial effects on the HSC pool, which have led to clinical trials of PTH treatment to enhance HSC-based therapies.

Haematopoietic stem cells depend on Galpha(s)-mediated signalling to engraft bone marrow.

Haematopoietic stem and progenitor cells (HSPCs) change location during development and circulate in mammals throughout life, moving into and out of the bloodstream to engage bone marrow niches in sequential steps of homing, engraftment and retention. Here we show that HSPC engraftment of bone marrow in fetal development is dependent on the guanine-nucleotide-binding protein stimulatory alpha subunit (Galpha(s)). HSPCs from adult mice deficient in Galpha(s) (Galpha(s)(-/-)) differentiate and undergo chemotaxis, but also do not home to or engraft in the bone marrow in adult mice and demonstrate a marked inability to engage the marrow microvasculature. If deleted after engraftment, Galpha(s) deficiency did not lead to lack of retention in the marrow, rather cytokine-induced mobilization into the blood was impaired. Testing whether activation of Galpha(s) affects HSPCs, pharmacological activators enhanced homing and engraftment in vivo. Galpha(s) governs specific aspects of HSPC localization under physiological conditions in vivo and may be pharmacologically targeted to improve transplantation efficiency.

Myeloproliferative disorder by TKO.

In this issue of Cell Stem Cell, Viatour et al. (2008) delete all three members of the retinoblastoma tumor suppressor gene family in hematopoietic stem cells, resulting in a myeloproliferative disorder. The disease was cell autonomous and resulted from alterations in the primitive hematopoietic cells.

Nf2/merlin regulates hematopoietic stem cell behavior by altering microenvironmental architecture.

Stem cell population size is highly regulated across species and tissue types, and alterations are associated with premature tissue failure or cancer. We assessed whether the tumor suppressor and mediator of cell contact inhibition Nf2/merlin plays a role in governing the hematopoietic stem cell pool by stem cell-autonomous or niche-determined processes. Hematopoietic stem cells in Nf2-deficient mice were increased in number and demonstrated a marked shift in location to the circulation. These changes were entirely dependent on changes in the microenvironment, with a marked increase in trabecular bone and marrow vascularity associated with increased VEGF, but without cell-autonomous alterations in stem cell characteristics. Nf2/merlin is critical for maintaining normal structure and function of the hematopoietic stem cell niche. It limits both bone and vascular components, and our model suggests that it thereby constrains stem cell number and position.

Deconstructing the hematopoietic stem cell niche: revealing the therapeutic potential.

Development of the hematopoietic system is a stage-specific process where the bone marrow eventually becomes the principal source of hematopoiesis in the adult mammalian organism. Sustained hematopoiesis in the bone marrow, however, depends on the self-renewal of the resident hematopoietic stem cells (HSCs). The region where these HSCs are hypothesized to self renew is called the stem cell 'niche.' Recent studies have identified components of the HSC niche in the bone marrow, including cells of the osteoblastic lineage, extracellular matrix molecules and molecular signaling interactions between the stem cells and niche cells. Specific pharmacological targeting of these niche components has led to beneficial HSC effects, demonstrating a new therapeutic approach where stem cell function is altered through targeting of the niche.

Therapeutic targeting of a stem cell niche.

The specialized microenvironment or niche where stem cells reside provides regulatory input governing stem cell function. We tested the hypothesis that targeting the niche might improve stem cell-based therapies using three mouse models that are relevant to clinical uses of hematopoietic stem (HS) cells. We and others previously identified the osteoblast as a component of the adult HS cell niche and established that activation of the parathyroid hormone (PTH) receptor on osteoblasts increases stem cell number. Here we show that pharmacologic use of PTH increases the number of HS cells mobilized into the peripheral blood for stem cell harvests, protects stem cells from repeated exposure to cytotoxic chemotherapy and expands stem cells in transplant recipients. These data provide evidence that the niche may be an attractive target for drug-based stem cell therapeutics.

The hematopoietic stem cell in its place.

A signature characteristic of stem cells is their ability to self-renew, affording a theoretically limitless ability to produce daughter cells and their descendents. This near-timeless dimension of stem cell function is not free of the constraints of place. The idea that highly specialized 'microenvironmental' cues participate in the regulation of stem cells has evidence in classic embryology and more recently in adult stem cells through the use of model organisms. There is now ample evidence that an anatomically defined, specifically constituted place represents the niche for hematopoietic and other tissue-specific stem cells. This review provides a conceptual framework and detailed account of the hematopoietic stem cell niche as defined at present. The components are assembling into a more complex view of the niche and may now be amenable to examination as a system and possibly to alteration to affect outcomes in immune regeneration.