Multispecific targeting of glioblastoma with tumor microenvironment-responsive multifunctional engineered NK cells.

Tumor antigen heterogeneity, a severely immunosuppressive tumor microenvironment (TME) and lymphopenia resulting in inadequate immune intratumoral trafficking, have rendered glioblastoma (GBM) highly resistant to therapy. To address these obstacles, here we describe a unique, sophisticated combinatorial platform for GBM: a cooperative multifunctional immunotherapy based on genetically engineered human natural killer (NK) cells bearing multiple antitumor functions including local tumor responsiveness that addresses key drivers of GBM resistance to therapy: antigen escape, immunometabolic reprogramming of immune responses, and poor immune cell homing. We engineered dual-specific chimeric antigen receptor (CAR) NK cells to bear a third functional moiety that is activated in the GBM TME and addresses immunometabolic suppression of NK cell function: a tumor-specific, locally released antibody fragment which can inhibit the activity of CD73 independently of CAR signaling and decrease the local concentration of adenosine. The multifunctional human NK cells targeted patient-derived GBM xenografts, demonstrated local tumor site-specific activity in the tissue, and potently suppressed adenosine production. We also unveil a complex reorganization of the immunological profile of GBM induced by inhibiting autophagy. Pharmacologic impairment of the autophagic process not only sensitized GBM to antigenic targeting by NK cells but promoted a chemotactic profile favorable to NK infiltration. Taken together, our study demonstrates a promising NK cell-based combinatorial strategy that can target multiple clinically recognized mechanisms of GBM progression simultaneously.

The neurotransmitter receptor Gabbr1 regulates proliferation and function of hematopoietic stem and progenitor cells.

Hematopoietic and nervous systems are linked via innervation of bone marrow (BM) niche cells. Hematopoietic stem/progenitor cells (HSPCs) express neurotransmitter receptors, such as the γ-aminobutyric acid (GABA) type B receptor subunit 1 (GABBR1), suggesting that HSPCs could be directly regulated by neurotransmitters like GABA that directly bind to GABBR1. We performed imaging mass spectrometry and found that the endogenous GABA molecule is regionally localized and concentrated near the endosteum of the BM niche. To better understand the role of GABBR1 in regulating HSPCs, we generated a constitutive Gabbr1-knockout mouse model. Analysis revealed that HSPC numbers were significantly reduced in the BM compared with wild-type littermates. Moreover, Gabbr1-null hematopoietic stem cells had diminished capacity to reconstitute irradiated recipients in a competitive transplantation model. Gabbr1-null HSPCs were less proliferative under steady-state conditions and upon stress. Colony-forming unit assays demonstrated that almost all Gabbr1-null HSPCs were in a slow or noncycling state. In vitro differentiation of Gabbr1-null HSPCs in cocultures produced fewer overall cell numbers with significant defects in differentiation and expansion of the B-cell lineage. To determine whether a GABBR1 agonist could stimulate human umbilical cord blood (UCB) HSPCs, we performed brief ex vivo treatment prior to transplant into immunodeficient mice, with significant increases in long-term engraftment of HSPCs compared with GABBR1 antagonist or vehicle treatments. Our results indicate a direct role for GABBR1 in HSPC proliferation, and identify a potential target to improve HSPC engraftment in clinical transplantation.

Bone marrow transplantation as a therapy for autosomal dominant osteopetrosis type 2 in mice.

Autosomal dominant osteopetrosis type II (ADO2) is a heritable bone disease of impaired osteoclastic bone resorption caused by missense mutations in the chloride channel 7 (CLCN7) gene. Clinical features of ADO2 include fractures, osteomyelitis of jaw, vision loss, and in severe cases, bone marrow failure. Currently, there is no effective therapy for ADO2, and patients usually receive symptomatic treatments. Theoretically, bone marrow transplantation (BMT), which is commonly used in recessive osteopetrosis, could be used to treat ADO2, although the frequency of complications related to BMT is quite high. We created an ADO2 knock-in (p.G213R mutation) mouse model on the 129 genetic background, and their phenotypes mimic the human disease of ADO2. To test whether BMT could restore osteoclast function and rescue the bone phenotypes in ADO2 mice, we transplanted bone marrow cells from 6-8 weeks old male WT donor mice into recipient female ADO2 mice. Also, to determine whether age at the time of transplant may play a role in transplant success, we performed BMT in young (12-week-old) and old (9-month-old) ADO2 mice. Our data indicate that ADO2 mice transplanted with WT marrow achieved more than 90% engraftment up to 6 months post-transplantation at both young and old ages. The in-vivo DXA data revealed that young ADO2 mice transplanted with WT marrow had significantly lower whole body and spine areal bone mineral density (aBMD) at month 6 post-transplantation compared to the ADO2 control mice. The old ADO2 mice also displayed significantly lower whole body, femur, and spine aBMD at months 4 and 5 post-transplantation compared to the age-matched control mice. The in-vivo micro-CT data showed that ADO2 experimental mice transplanted with WT marrow had significantly lower BV/TV at months 2 and 4 post-transplantation compared to the ADO2 control mice at a young age. In contrast, ADO2 control and experimental mice displayed similar BV/TV values for all post-transplantation time points at old age. In addition, serum CTX was significantly higher at month 2 post-transplantation in both young and old ADO2 experimental mice compared to the ADO2 control mice. Serum P1NP levels in young ADO2 experimental mice were significantly higher at baseline and month 2 post-transplantation compared to the ADO2 control mice. These data suggest that BMT may provide, at least, some beneficial effect at both young and adult ages.

Characterization and initial demonstration of in vivo efficacy of a novel heat-activated metalloenediyne anti-cancer agent.

BACKGROUND: Enediynes are anti-cancer agents that are highly cytotoxic due to their propensity for low thermal activation of radical generation. The diradical intermediate produced from Bergman cyclization of the enediyne moiety may induce DNA damage and cell lethality. The cytotoxicity of enediynes and difficulties in controlling their thermal cyclization has limited their clinical use. We recently showed that enediyne toxicity at 37 °C can be mitigated by metallation, but cytotoxic effects of 'metalloenediynes' on cultured tumor cells are potentiated by hyperthermia. Reduction of cytotoxicity at normothermia suggests metalloenediynes will have a large therapeutic margin, with cell death occurring primarily in the heated tumor. Based on our previous in vitro findings, FeSO4-PyED, an Fe co-factor complex of (Z)-N,N'-bis[1-pyridin-2-yl-meth-(E)-ylidene]oct-4-ene-2,6-diyne-1,8-diamine, was prioritized for further in vitro and in vivo testing in normal human melanocytes and melanoma cells. METHODS: Clonogenic survival, apopotosis and DNA binding assays were used to determine mechanisms of enhancement of FeSO4-PyED cytotoxicity by hyperthermia. A murine human melanoma xenograft model was used to assess in vivo efficacy of FeSO4-PyED at 37 or 42.5 °C. RESULTS: FeSO4-PyED is a DNA-binding compound. Enhancement of FeSO4-PyED cytotoxicity by hyperthermia in melanoma cells was due to Bergman cyclization, diradical formation, and increased apoptosis. Thermal enhancement, however, was not observed in melanocytes. FeSO4-PyED inhibited tumor growth when melanomas were heated during drug treatment, without inducing normal tissue damage. CONCLUSION: By leveraging the unique thermal activation properties of metalloenediynes, we propose that localized moderate hyperthermia can be used to confine the cytotoxicity of these compounds to tumors, while sparing normal tissue.

A heterobivalent ligand inhibits mast cell degranulation via selective inhibition of allergen-IgE interactions in vivo.

Current treatments for allergies include epinephrine and antihistamines, which treat the symptoms after an allergic response has taken place; steroids, which result in local and systemic immune suppression; and IgE-depleting therapies, which can be used only for a narrow range of clinical IgE titers. The limitations of current treatments motivated the design of a heterobivalent inhibitor (HBI) of IgE-mediated allergic responses that selectively inhibits allergen-IgE interactions, thereby preventing IgE clustering and mast cell degranulation. The HBI was designed to simultaneously target the allergen binding site and the adjacent conserved nucleotide binding site (NBS) found on the Fab of IgE Abs. The bivalent targeting was accomplished by linking a hapten to an NBS ligand with an ethylene glycol linker. The hapten moiety of HBI enables selective targeting of a specific IgE, whereas the NBS ligand enhances avidity for the IgE. Simultaneous bivalent binding to both sites provided HBI with 120-fold enhancement in avidity for the target IgE compared with the monovalent hapten. The increased avidity for IgE made HBI a potent inhibitor of mast cell degranulation in the rat basophilic leukemia mast cell model, in the passive cutaneous anaphylaxis mouse model of allergy, and in mice sensitized to the model allergen. In addition, HBI did not have any observable systemic toxic effects even at elevated doses. Taken together, these results establish the HBI design as a broadly applicable platform with therapeutic potential for the targeted and selective inhibition of IgE-mediated allergic responses, including food, environmental, and drug allergies.

Soypeptide lunasin in cytokine immunotherapy for lymphoma.

Immunostimulatory cytokines can enhance anti-tumor immunity and are part of the therapeutic armamentarium for cancer treatment. We have previously reported that post-transplant lymphoma patients have an acquired deficiency of signal transducer and activator of transcription 4, which results in defective IFNγ production during clinical immunotherapy. With the goal of further improving cytokine-based immunotherapy, we examined the effects of a soybean peptide called lunasin that synergistically works with cytokines on natural killer (NK) cells. Peripheral blood mononuclear cells of healthy donors and post-transplant lymphoma patients were stimulated with or without lunasin in the presence of IL-12 or IL-2. NK activation was evaluated, and its tumoricidal activity was assessed using in vitro and in vivo tumor models. Chromatin immunoprecipitation assay was performed to evaluate the histone modification of gene loci that are regulated by lunasin and cytokine. Adding lunasin to IL-12- or IL-2-stimulated NK cells demonstrated synergistic effects in the induction of IFNG and GZMB involved in cytotoxicity. The combination of lunasin and cytokines (IL-12 plus IL-2) was capable of restoring IFNγ production by NK cells from post-transplant lymphoma patients. In addition, NK cells stimulated with lunasin plus cytokines displayed higher tumoricidal activity than those stimulated with cytokines alone using in vitro and in vivo tumor models. The underlying mechanism responsible for the effects of lunasin on NK cells is likely due to epigenetic modulation on target gene loci. Lunasin represents a different class of immune modulating agent that may augment the therapeutic responses mediated by cytokine-based immunotherapy.

Human proangiogenic circulating hematopoietic stem and progenitor cells promote tumor growth in an orthotopic melanoma xenograft model.

We previously identified a distinct population of human circulating hematopoietic stem and progenitor cells (CHSPCs; CD14(-)glyA(-)CD34(+)AC133(+/-)CD45(dim)CD31(+) cells) in the peripheral blood (PB) and bone marrow, and their frequency in the PB can correlate with disease state. The proangiogenic subset (pCHSPC) play a role in regulating tumor progression, for we previously demonstrated a statistically significant increase in C32 melanoma growth in NOD.Cg-Prkdc (scid) (NOD/SCID) injected with human pCHSPCs (p < 0.001). We now provide further evidence that pCHSPCs possess proangiogenic properties. In vitro bio-plex cytokine analyses and tube forming assays indicate that pCHSPCs secrete a proangiogenic profile and promote vessel formation respectively. We also developed a humanized bone marrow-melanoma orthotopic model to explore in vivo the biological significance of the pCHSPC population. Growth of melanoma xenografts increased more rapidly at 3-4 weeks post-tumor implantation in mice previously transplanted with human CD34(+) cells compared to control mice. Increases in pCHSPCs in PB correlated with increases in tumor growth. Additionally, to determine if we could prevent the appearance of pCHSPCs in the PB, mice with humanized bone marrow-melanoma xenografts were administered Interferon α-2b, which is used clinically for treatment of melanoma. The mobilization of the pCHSPCs was decreased in the mice with the humanized bone marrow-melanoma xenografts. Taken together, these data indicate that pCHSPCs play a functional role in tumor growth. The novel in vivo model described here can be utilized to further validate pCHSPCs as a biomarker of tumor progression. The model can also be used to screen and optimize anticancer/anti-angiogenic therapies in a humanized system.

Small-molecule inhibition of the uPAR·uPA interaction: synthesis, biochemical, cellular, in vivo pharmacokinetics and efficacy studies in breast cancer metastasis.

The uPAR·uPA protein-protein interaction (PPI) is involved in signaling and proteolytic events that promote tumor invasion and metastasis. A previous study had identified 4 (IPR-803) from computational screening of a commercial chemical library and shown that the compound inhibited uPAR·uPA PPI in competition biochemical assays and invasion cellular studies. Here, we synthesize 4 to evaluate in vivo pharmacokinetic (PK) and efficacy studies in a murine breast cancer metastasis model. First, we show, using fluorescence polarization and saturation transfer difference (STD) NMR, that 4 binds directly to uPAR with sub-micromolar affinity of 0.2 µM. We show that 4 blocks invasion of breast MDA-MB-231, and inhibits matrix metalloproteinase (MMP) breakdown of the extracellular matrix (ECM). Derivatives of 4 also inhibited MMP activity and blocked invasion in a concentration-dependent manner. Compound 4 also impaired MDA-MB-231 cell adhesion and migration. Extensive in vivo PK studies in NOD-SCID mice revealed a half-life of nearly 5h and peak concentration of 5 µM. Similar levels of the inhibitor were detected in tumor tissue up to 10h. Female NSG mice inoculated with highly malignant TMD-MDA-MB-231 in their mammary fat pads showed that 4 impaired metastasis to the lungs with only four of the treated mice showing severe or marked metastasis compared to ten for the untreated mice. Compound 4 is a promising template for the development of compounds with enhanced PK parameters and greater efficacy.

Characterization of the Stereoselective Disposition of Bupropion and Its Metabolites in Rat Plasma and Brain.

BACKGROUND AND OBJECTIVES: Bupropion is an atypical antidepressant and smoking cessation aid; its use is associated with wide intersubject variability in efficacy and safety. Knowledge of the brain pharmacokinetics of bupropion and its pharmacologically active metabolites is considered important for understanding the cause-effect relationships driving this variability. METHODS: Brain concentrations from rats administered a 10 mg/kg subcutaneous dose of racemic bupropion were analyzed using a stereoselective LC/MS-MS method. A 2 mg/kg dose of (S,S)-hydroxybupropion, which has comparable pharmacologic potency to bupropion, was administered to a separate group of rats. Plasma exposure and unbound concentrations in both matrices from companion equilibrium dialysis experiments were determined to assess potential carrier-mediated transport at the blood-brain barrier. RESULTS: Exposures to unbound forms of bupropion enantiomers were similar in plasma; this was also true in brain. This trend held for reductive diastereomer metabolite pairs in the two matrices. Unbound (R,R)-hydroxybupropion exposure was 1.5-fold higher than (S,S)-hydroxybupropion exposure in plasma and brain following bupropion administration. Unbound concentration ratios (Kp,uu) of a given molecular form decreased over time: between 4 and 6 h, these were < 1 for the two bupropion enantiomers, and they were ~ 1 for metabolites that formed. Administration of preformed (S,S)-hydroxybupropion also demonstrated a declining Kp,uu. CONCLUSIONS: The temporal shift in Kp,uu among the different molecular forms provides evidence regarding the operation of carrier-mediated transport and/or within-brain metabolism of bupropion, and, thereby, fresh insight regarding the causes of intersubject variability in the safety and efficacy of bupropion therapy.

Peanut allergen inhibition prevents anaphylaxis in a humanized mouse model.

Peanut-induced allergy is an immunoglobulin E (IgE)-mediated type I hypersensitivity reaction that manifests symptoms ranging from local edema to life-threatening anaphylaxis. Although there are treatments for symptoms in patients with allergies resulting from allergen exposure, there are few preventive therapies other than strict dietary avoidance or oral immunotherapy, neither of which are successful in all patients. We have previously designed a covalent heterobivalent inhibitor (cHBI) that binds in an allergen-specific manner as a preventive for allergic reactions. Building on previous in vitro testing, here, we developed a humanized mouse model to test cHBI efficacy in vivo. Nonobese diabetic-severe combined immunodeficient γc-deficient mice expressing transgenes for human stem cell factor, granulocyte-macrophage colony-stimulating factor, and interleukin-3 developed mature functional human mast cells in multiple tissues and displayed robust anaphylactic reactions when passively sensitized with patient-derived IgE monoclonal antibodies specific for peanut Arachis hypogaea 2 (Ara h 2). The allergic response in humanized mice was IgE dose dependent and was mediated by human mast cells. Using this humanized mouse model, we showed that cHBI prevented allergic reactions for more than 2 weeks when administered before allergen exposure. cHBI also prevented fatal anaphylaxis and attenuated allergic reactions when administered shortly after the onset of symptoms. cHBI impaired mast cell degranulation in vivo in an allergen-specific manner. cHBI rescued the mice from lethal anaphylactic responses during oral Ara h 2 allergen-induced anaphylaxis. Together, these findings suggest that cHBI has the potential to be an effective preventative for peanut-specific allergic responses in patients.

Characterization and Function of Cryopreserved Bone Marrow from Deceased Organ Donors: A Potential Viable Alternative Graft Source.

Despite the readily available graft sources for allogeneic hematopoietic cell transplantation (alloHCT), a significant unmet need remains in the timely provision of suitable unrelated donor grafts. This shortage is related to the rarity of certain HLA alleles in the donor pool, nonclearance of donors owing to infectious disease or general health status, and prolonged graft procurement and processing times. An alternative hematopoietic progenitor cell (HPC) graft source obtained from the vertebral bodies (VBs) of deceased organ donors could alleviate many of the obstacles associated with using grafts from healthy living donors or umbilical cord blood (UCB). Deceased organ donor-derived bone marrow (BM) can be preemptively screened, cryogenically banked for on-demand use, and made available in adequate cell doses for HCT. We have developed a good manufacturing practice (GMP)-compliant process to recover and cryogenically bank VB-derived HPCs from deceased organ donor (OD) BM. Here we present results from an analysis of HPCs from BM obtained from 250 deceased donors to identify any substantial difference in composition or quality compared with HPCs from BM aspirated from the iliac crests of healthy living donors. BM from deceased donor VBs was processed in a central GMP facility and packaged for cryopreservation in 5% DMSO/2.5% human serum albumin. BM aspirated from living donor iliac crests was obtained and used for comparison. A portion of each specimen was analyzed before and after cryopreservation by flow cytometry and colony-forming unit potential. Bone marrow chimerism potential was assessed in irradiated immunocompromised NSG mice. Analysis of variance with Bonferroni correction for multiple comparisons was used to determine how cryopreservation affects BM cells and to evaluate indicators of successful engraftment of BM cells into irradiated murine models. The t test (with 95% confidence intervals [CIs]) was used to compare cells from deceased donors and living donors. A final dataset of complete clinical and matched laboratory data from 226 cryopreserved samples was used in linear regressions to predict outcomes of BM HPC processing. When compared before and after cryopreservation, OD-derived BM HPCs were found to be stable, with CD34+ cells maintaining high viability and function after thawing. The yield from a single donor is sufficient for transplantation of an average of 1.6 patients (range, 1.2 to 7.5). CD34+ cells from OD-derived HPCs from BM productively engrafted sublethally irradiated immunocompromised mouse BM (>44% and >67% chimerism at 8 and 16 weeks, respectively). Flow cytometry and secondary transplantation confirmed that OD HPCs from BM is composed of long-term engrafting CD34+CD38-CD45RA-CD90+CD49f+ HSCs. Linear regression identified no meaningful predictive associations between selected donor-related characteristics and OD BM HPC quality or yield. Collectively, these data demonstrate that cryopreserved BM HPCs from deceased organ donors is potent and functionally equivalent to living donor BM HPCs and is a viable on-demand graft source for clinical HCT. Prospective clinical trials will soon commence in collaboration with the Center for International Blood and Marrow Research to assess the feasibility, safety, and efficacy of Ossium HPCs from BM (ClinicalTrials.gov identifier NCT05068401).

Establishment and characterization of patient-derived xenograft of a rare pediatric anaplastic pleomorphic xanthoastrocytoma (PXA) bearing a CDC42SE2-BRAF fusion.

Pleomorphic xanthoastrocytoma (PXA) is a rare subset of primary pediatric glioma with 70% 5-year disease free survival. However, up to 20% of cases present with local recurrence and malignant transformation into more aggressive type anaplastic PXA (AXPA) or glioblastoma. The understanding of disease etiology and mechanisms driving PXA and APXA are limited, and there is no standard of care. Therefore, development of relevant preclinical models to investigate molecular underpinnings of disease and to guide novel therapeutic approaches are of interest. Here, for the first time we established, and characterized a patient-derived xenograft (PDX) from a leptomeningeal spread of a patient with recurrent APXA bearing a novel CDC42SE2-BRAF fusion. An integrated -omics analysis was conducted to assess model fidelity of the genomic, transcriptomic, and proteomic/phosphoproteomic landscapes. A stable xenoline was derived directly from the patient recurrent tumor and maintained in 2D and 3D culture systems. Conserved histology features between the PDX and matched APXA specimen were maintained through serial passages. Whole exome sequencing (WES) demonstrated a high degree of conservation in the genomic landscape between PDX and matched human tumor, including small variants (Pearson's r = 0.794-0.839) and tumor mutational burden (~ 3 mutations/MB). Large chromosomal variations including chromosomal gains and losses were preserved in PDX. Notably, chromosomal gain in chromosomes 4-9, 17 and 18 and loss in the short arm of chromosome 9 associated with homozygous 9p21.3 deletion involving CDKN2A/B locus were identified in both patient tumor and PDX sample. Moreover, chromosomal rearrangement involving 7q34 fusion; CDC42SE-BRAF t (5;7) (q31.1, q34) (5:130,721,239, 7:140,482,820) was identified in the PDX tumor, xenoline and matched human tumor. Transcriptomic profile of the patient's tumor was retained in PDX (Pearson r = 0.88) and in xenoline (Pearson r = 0.63) as well as preservation of enriched signaling pathways (FDR Adjusted P < 0.05) including MAPK, EGFR and PI3K/AKT pathways. The multi-omics data of (WES, transcriptome, and reverse phase protein array (RPPA) was integrated to deduce potential actionable pathways for treatment (FDR < 0.05) including KEGG01521, KEGG05202, and KEGG05200. Both xenoline and PDX were resistant to the MEK inhibitors trametinib or mirdametinib at clinically relevant doses, recapitulating the patient's resistance to such treatment in the clinic. This set of APXA models will serve as a preclinical resource for developing novel therapeutic regimens for rare anaplastic PXAs and pediatric high-grade gliomas bearing BRAF fusions.

Integrative Multi-OMICs Identifies Therapeutic Response Biomarkers and Confirms Fidelity of Clinically Annotated, Serially Passaged Patient-Derived Xenografts Established from Primary and Metastatic Pediatric and AYA Solid Tumors.

Establishment of clinically annotated, molecularly characterized, patient-derived xenografts (PDXs) from treatment-naïve and pretreated patients provides a platform to test precision genomics-guided therapies. An integrated multi-OMICS pipeline was developed to identify cancer-associated pathways and evaluate stability of molecular signatures in a panel of pediatric and AYA PDXs following serial passaging in mice. Original solid tumor samples and their corresponding PDXs were evaluated by whole-genome sequencing, RNA-seq, immunoblotting, pathway enrichment analyses, and the drug−gene interaction database to identify as well as cross-validate actionable targets in patients with sarcomas or Wilms tumors. While some divergence between original tumor and the respective PDX was evident, majority of alterations were not functionally impactful, and oncogenic pathway activation was maintained following serial passaging. CDK4/6 and BETs were prioritized as biomarkers of therapeutic response in osteosarcoma PDXs with pertinent molecular signatures. Inhibition of CDK4/6 or BETs decreased osteosarcoma PDX growth (two-way ANOVA, p < 0.05) confirming mechanistic involvement in growth. Linking patient treatment history with molecular and efficacy data in PDX will provide a strong rationale for targeted therapy and improve our understanding of which therapy is most beneficial in patients at diagnosis and in those already exposed to therapy.

Leptin receptor, a surface marker for a subset of highly engrafting long-term functional hematopoietic stem cells.

The hematopoietic system is sustained by a rare population of hematopoietic stem cells (HSCs), which emerge during early embryonic development and then reside in the hypoxic niche of the adult bone marrow microenvironment. Although leptin receptor (Lepr)-expressing stromal cells are well-studied as critical regulators of murine hematopoiesis, the biological implications of Lepr expression on HSCs remain largely unexplored. We hypothesized that Lepr+HSCs are functionally different from other HSCs. Using in vitro and in vivo experimental approaches, we demonstrated that Lepr further differentiates SLAM HSCs into two distinct populations; Lepr+HSCs engrafted better than Lepr-HSCs in primary transplant. Compared to Lepr-LSK cells, Lepr+LSK cells were highly enriched for extensively repopulating and self-renewing HSCs. Molecularly, Lepr+HSCs were characterized by a pro-inflammatory transcriptomic profile enriched for Type-I Interferon and Interferon-gamma (IFN-γ) response pathways, which are known to be critical for the emergence of HSCs in the embryo. We conclude that although Lepr+HSCs represent a minor subset of HSCs, they are highly engrafting cells that possess embryonic-like transcriptomic characteristics, and that Lepr can serve as a reliable marker for functional long-term HSCs, which may have potential clinical applicability.

Preclinical activity of a novel multiple tyrosine kinase and aurora kinase inhibitor, ENMD-2076, against multiple myeloma.

ENMD-2076 is a novel, orally-active molecule that has been shown to have significant activity against aurora and multiple receptor tyrosine kinases. We investigated the activity of ENMD-2076 against multiple myeloma (MM) cells in vitro and in vivo. ENMD-2076 showed significant cytotoxicity against MM cell lines and primary cells, with minimal cytotoxicity to haematopoietic progenitors. ENMD-2076 inhibited the phosphoinositide 3-kinase/AKT pathway and downregulated survivin and X-linked inhibitor of apoptosis as early as 6 h after treatment. With longer treatment (24-48 h), ENMD-2076 also inhibited aurora A and B kinases, and induced G(2)/M cell cycle arrest. In non-obese diabetic/severe combined immunodeficient mice implanted with H929 human plasmacytoma xenografts, oral treatment with ENMD-2076 (50, 100, 200 mg/kg per day) resulted in a dose-dependent inhibition of tumour growth. Immunohistochemical staining of excised tumours showed significant reduction in phospho-Histone 3 (pH3), Ki-67, and angiogenesis, and also a significant increase in cleaved caspase-3 at all dose levels compared to tumours from vehicle-treated mice. In addition, a significant reduction in p-FGFR3 was observed on Western blot. ENMD-2076 shows significant activity against MM cells in vitro and in vivo, and acts on several pathways important for myeloma cell growth and survival. These results provide preclinical rationale for clinical investigation of ENMD-2076 in MM.

How old is too old? In vivo engraftment of human peripheral blood stem cells cryopreserved for up to 18 years - implications for clinical transplantation and stability programs.

BACKGROUND: Peripheral blood stem cells (PBSC) are commonly cryopreserved awaiting clinical use for hematopoietic stem cell transplant. Long term cryopreservation is commonly defined as five years or longer, and limited data exists regarding how long PBSC can be cryopreserved and retain the ability to successfully engraft. Clinical programs, stem cell banks, and regulatory and accrediting agencies interested in product stability would benefit from such data. Thus, we assessed recovery and colony forming ability of PBSC following long-term cryopreservation as well as their ability to engraft in NOD/SCID/IL-2Rγnull (NSG) mice. AIM: To investigate the in vivo engraftment potential of long-term cryopreserved PBSC units. METHODS: PBSC units which were collected and frozen using validated clinical protocols were obtained for research use from the Cellular Therapy Laboratory at Indiana University Health. These units were thawed in the Cellular Therapy Laboratory using clinical standards of practice, and the pre-freeze and post-thaw characteristics of the units were compared. Progenitor function was assessed using standard colony-forming assays. CD34-selected cells were transplanted into immunodeficient mice to assess stem cell function. RESULTS: Ten PBSC units with mean of 17 years in cryopreservation (range 13.6-18.3 years) demonstrated a mean total cell recovery of 88% ± 12% (range 68%-110%) and post-thaw viability of 69% ± 17% (range 34%-86%). BFU-E growth was shown in 9 of 10 units and CFU-GM growth in 7 of 10 units post-thaw. Immunodeficient mice were transplanted with CD34-selected cells from four randomly chosen PBSC units. All mice demonstrated long-term engraftment at 12 wk with mean 34% ± 24% human CD45+ cells, and differentiation with presence of human CD19+, CD3+ and CD33+ cells. Harvested bone marrow from all mice demonstrated growth of erythroid and myeloid colonies. CONCLUSION: We demonstrated engraftment of clinically-collected and thawed PBSC following cryopreservation up to 18 years in NSG mice, signifying likely successful clinical transplantation of PBSC following long-term cryopreservation.

Systems Biology Approach Identifies Prognostic Signatures of Poor Overall Survival and Guides the Prioritization of Novel BET-CHK1 Combination Therapy for Osteosarcoma.

Osteosarcoma (OS) patients exhibit poor overall survival, partly due to copy number variations (CNVs) resulting in dysregulated gene expression and therapeutic resistance. To identify actionable prognostic signatures of poor overall survival, we employed a systems biology approach using public databases to integrate CNVs, gene expression, and survival outcomes in pediatric, adolescent, and young adult OS patients. Chromosome 8 was a hotspot for poor prognostic signatures. The MYC-RAD21 copy number gain (8q24) correlated with increased gene expression and poor overall survival in 90% of the patients (n = 85). MYC and RAD21 play a role in replication-stress, which is a therapeutically actionable network. We prioritized replication-stress regulators, bromodomain and extra-terminal proteins (BETs), and CHK1, in order to test the hypothesis that the inhibition of BET + CHK1 in MYC-RAD21+ pediatric OS models would be efficacious and safe. We demonstrate that MYC-RAD21+ pediatric OS cell lines were sensitive to the inhibition of BET (BETi) and CHK1 (CHK1i) at clinically achievable concentrations. While the potentiation of CHK1i-mediated effects by BETi was BET-BRD4-dependent, MYC expression was BET-BRD4-independent. In MYC-RAD21+ pediatric OS xenografts, BETi + CHK1i significantly decreased tumor growth, increased survival, and was well tolerated. Therefore, targeting replication stress is a promising strategy to pursue as a therapeutic option for this devastating disease.

A novel competitive repopulation strategy to quantitate engraftment of ex vivo manipulated murine marrow cells in submyeloablated hosts.

OBJECTIVE: Standard competitive repopulation assays have proven valuable in evaluating engraftment potential in ablated hosts, permitting comparisons between various test cell populations. However, no similar method exists to compare engraftment of test cells in submyeloablated hosts, which would be helpful given the applications of reduced-intensity conditioning for hematopoietic gene-replacement therapy and other cellular therapies. Here, we developed a novel assay to quantitate engraftment of hematopoietic stem cells in submyeloablated hosts. MATERIALS AND METHODS: Engraftment of murine marrow cells transduced with retroviral vectors using two separate protocols was compared to engraftment of fresh untreated competitor cells within low-dose radiation-conditioned hosts using a "three-way" marking system, so that test, competitor, and host cell chimerism could be reliably determined posttransplantation. RESULTS: We demonstrate that the repopulating ability of marrow cells transduced using two distinct protocols was reduced approximately 10-fold compared to fresh competitor cells in submyeloablated hosts utilizing the novel "three-way" transplant assay. CONCLUSIONS: Murine marrow cells transduced using a clinically applicable protocol acquire an engraftment defect in submyeloablated hosts, similar to cells transduced using a research protocol. We conclude that the submyeloablative competitive repopulation assay described here will be of benefit to comparatively assess the engraftment ability of manipulated hematopoietic stem cells using various culture protocols, such as to test the impact of modifications in transduction protocols needed to attain therapeutic levels of gene-corrected blood cells, or the effect of ex vivo expansion protocols on engraftment potential.

In vivo selection of hematopoietic stem cells transduced at a low multiplicity-of-infection with a foamy viral MGMT(P140K) vector.

OBJECTIVE: Using a clinically relevant transduction strategy, we investigated to what extent hematopoietic stem cells in lineage-negative bone marrow (Lin(neg) BM) could be genetically modified with an foamy virus (FV) vector that expresses the DNA repair protein, O(6)-methylguanine DNA methyltransferase (MGMT(P140K)) and selected in vivo with submyeloablative or myeloablative alkylator therapy. MATERIALS AND METHODS: Lin(neg) BM was transduced at a low multiplicity-of-infection with the FV vector, MD9-P140K, which coexpresses MGMT(P140K) and the enhanced green fluorescent protein, transplanted into C57BL/6 mice, and mice treated with submyeloablative or myeloablative alkylator therapy. The BM was analyzed for the presence of in vivo selected, MD9-P140K-transduced cells at 6 months post-transplantation and subsequently transplanted into secondary recipient animals. RESULTS: Following submyeloablative therapy, 55% of the mice expressed MGMT(P140K) in the BM. Proviral integration was observed in approximately 50% of committed BM-derived progenitors and analysis of proviral insertion sites indicated up to two integrations per transduced progenitor colony. Transduced BM cells selected with submyeloablative therapy reconstituted secondary recipient mice for up to 6 months post-transplantation. In contrast, after delivery of myeloablative therapy to primary recipient mice, only 25% survived. Hematopoietic stem cells were transduced because BM cells from the surviving animals reconstituted secondary recipients with MGMT(P140K)-positive cells for 5 to 6 months. CONCLUSIONS: In vivo selection of MD9-P140K-transduced BM cells was more efficient following submyeloablative than myeloablative therapy. These data indicate that a critical number of transduced stem cells must be present to produce sufficient numbers of genetically modified progeny to protect against acute toxicity associated with myeloablative therapy.