Breast Cancer Mutations HER2V777L and PIK3CAH1047R Activate the p21-CDK4/6-Cyclin D1 Axis to Drive Tumorigenesis and Drug Resistance.

In metastatic breast cancer, HER2-activating mutations frequently co-occur with mutations in PIK3CA, TP53, or CDH1. Of these co-occurring mutations, HER2 and PIK3CA are the most commonly comutated gene pair, with approximately 40% of HER2-mutated breast cancers also having activating mutations in PIK3CA. To study the effects of co-occurring HER2 and PIK3CA mutations, we generated genetically engineered mice with the HER2V777L; PIK3CAH1047R transgenes (HP mice) and studied the resulting breast cancers both in vivo as well as ex vivo using cancer organoids. HP breast cancers showed accelerated tumor formation in vivo and increased invasion and migration in in vitro assays. HP breast cancer cells were resistant to the pan-HER tyrosine kinase inhibitor, neratinib, but were effectively treated with neratinib plus the HER2-targeted antibody-drug conjugate trastuzumab deruxtecan. Proteomic and RNA-seq analysis of HP breast cancers identified increased gene expression of cyclin D1 and p21WAF1/Cip1 and changes in cell-cycle markers. Combining neratinib with CDK4/6 inhibitors was another effective strategy for treating HP breast cancers, with neratinib plus palbociclib showing a statistically significant reduction in development of mouse HP tumors as compared to either drug alone. The efficacy of both the neratinib plus trastuzumab deruxtecan and neratinib plus palbociclib combinations was validated using a human breast cancer patient-derived xenograft with very similar HER2 and PIK3CA mutations to the HP mice. Further, these two drug combinations effectively treated spontaneous lung metastasis in syngeneic mice transplanted with HP breast cancer organoids. This study provides valuable preclinical data to support the ongoing phase 1 clinical trials of these drug combinations in breast cancer. SIGNIFICANCE: In HER2-mutated breast cancer, PIK3CA mutation activates p21-CDK4/6-cyclin D1 signaling to drive resistance to HER2-targeted therapies, which can be overcome using CDK4/6 inhibitors.

CS1 CAR-T targeting the distal domain of CS1 (SLAMF7) shows efficacy in high tumor burden myeloma model despite fratricide of CD8+CS1 expressing CAR-T cells.

Despite improvement in treatment options for myeloma patients, including targeted immunotherapies, multiple myeloma remains a mostly incurable malignancy. High CS1 (SLAMF7) expression on myeloma cells and limited expression on normal cells makes it a promising target for CAR-T therapy. The CS1 protein has two extracellular domains - the distal Variable (V) domain and the proximal Constant 2 (C2) domain. We generated and tested CS1-CAR-T targeting the V domain of CS1 (Luc90-CS1-CAR-T) and demonstrated anti-myeloma killing in vitro and in vivo using two mouse models. Since fratricide of CD8 + cells occurred during production, we generated fratricide resistant CS1 deficient Luc90- CS1- CAR-T (ΔCS1-Luc90- CS1- CAR-T). This led to protection of CD8 + cells in the CAR-T cultures, but had no impact on efficacy. Our data demonstrate targeting the distal V domain of CS1 could be an effective CAR-T treatment for myeloma patients and deletion of CS1 in clinical production did not provide an added benefit using in vivo immunodeficient NSG preclinical models.

Calcium carbonate nanoparticles stimulate tumor metabolic reprogramming and modulate tumor metastasis.

AIM: CaCO3 nanoparticles (nano-CaCO3) can neutralize the acidic pHe of solid tumors, but the lack of intrinsic imaging signal precludes noninvasive monitoring of pH-perturbation in tumor microenvironment. We aim to develop a theranostic version of nano-CaCO3 to noninvasively monitor pH modulation and subsequent tumor response. MATERIALS & METHODS: We synthesized ferromagnetic core coated with CaCO3 (magnetite CaCO3). Magnetic resonance imaging (MRI) was used to determine the biodistribution and pH modulation using murine fibrosarcoma and breast cancer models. RESULTS: Magnetite CaCO3-MRI imaging showed that nano-CaCO3 rapidly raised tumor pHe, followed by excessive tumor-associated acid production after its clearance. Continuous nano-CaCO3 infusion could inhibit metastasis. CONCLUSION: Nano-CaCO3 exposure induces tumor metabolic reprogramming that could account for the failure of previous intermittent pH-modulation strategies to achieve sustainable therapeutic effect.

An "off-the-shelf" fratricide-resistant CAR-T for the treatment of T cell hematologic malignancies.

T cell malignancies represent a group of hematologic cancers with high rates of relapse and mortality in patients for whom no effective targeted therapies exist. The shared expression of target antigens between chimeric antigen receptor (CAR) T cells and malignant T cells has limited the development of CAR-T because of unintended CAR-T fratricide and an inability to harvest sufficient autologous T cells. Here, we describe a fratricide-resistant "off-the-shelf" CAR-T (or UCART7) that targets CD7+ T cell malignancies and, through CRISPR/Cas9 gene editing, lacks both CD7 and T cell receptor alpha chain (TRAC) expression. UCART7 demonstrates efficacy against human T cell acute lymphoblastic leukemia (T-ALL) cell lines and primary T-ALL in vitro and in vivo without the induction of xenogeneic GvHD. Fratricide-resistant, allo-tolerant "off-the-shelf" CAR-T represents a strategy for treatment of relapsed and refractory T-ALL and non-Hodgkin's T cell lymphoma without a requirement for autologous T cells.

A new model of multi-visceral and bone metastatic prostate cancer with perivascular niche targeting by a novel endothelial specific adenoviral vector.

While modern therapies for metastatic prostate cancer (PCa) have improved survival they are associated with an increasingly prevalent entity, aggressive variant PCa (AVPCa), lacking androgen receptor (AR) expression, enriched for cancer stem cells (CSCs), and evidencing epithelial-mesenchymal plasticity with a varying extent of neuroendocrine transdifferentiation. Parallel work revealed that endothelial cells (ECs) create a perivascular CSC niche mediated by juxtacrine and membrane tethered signaling. There is increasing interest in pharmacological metastatic niche targeting, however, targeted access has been impossible. Here, we discovered that the Gleason 7 derived, androgen receptor negative, IGR-CaP1 cell line possessed some but not all of the molecular features of AVPCa. Intracardiac injection into NOD/SCID/IL2Rg -/- (NSG) mice produced a completely penetrant bone, liver, adrenal, and brain metastatic phenotype; noninvasively and histologically detectable at 2 weeks, and necessitating sacrifice 4-5 weeks post injection. Bone metastases were osteoblastic, and osteolytic. IGR-CaP1 cells expressed the neuroendocrine marker synaptophysin, near equivalent levels of vimentin and e-cadherin, all of the EMT transcription factors, and activation of NOTCH and WNT pathways. In parallel, we created a new triple-targeted adenoviral vector containing a fiber knob RGD peptide, a hexon mutation, and an EC specific ROBO4 promoter (Ad.RGD.H5/3.ROBO4). This vector was expressed in metastatic microvessels tightly juxtaposed to IGR-CaP1 cells in bone and visceral niches. Thus, the combination of IGR-CaP1 cells and NSG mice produces a completely penetrant metastatic PCa model emulating end-stage human disease. In addition, the metastatic niche access provided by our novel Ad vector could be therapeutically leveraged for future disease control or cure.

Fluselenamyl: A Novel Benzoselenazole Derivative for PET Detection of Amyloid Plaques (Aß) in Alzheimer's Disease.

Fluselenamyl (5), a novel planar benzoselenazole shows traits desirable of enabling noninvasive imaging of Aß pathophysiology in vivo; labeling of both diffuse (an earlier manifestation of neuritic plaques) and fibrillar plaques in Alzheimer's disease (AD) brain sections, and remarkable specificity for mapping Aß compared with biomarker proteins of other neurodegenerative diseases. Employing AD homogenates, [18F]-9, a PET tracer demonstrates superior (2-10 fold higher) binding affinity than approved FDA tracers, while also indicating binding to high affinity site on Aß plaques. Pharmacokinetic studies indicate high initial influx of [18F]-9 in normal mice brains accompanied by rapid clearance in the absence of targeted plaques. Following incubation in human serum, [18F]-9 indicates presence of parental compound up to 3h thus indicating its stability. Furthermore, in vitro autoradiography studies of [18F]-9 with AD brain tissue sections and ex vivo autoradiography studies in transgenic mouse brain sections show cortical Aß binding, and a fair correlation with Aß immunostaining. Finally, multiphoton- and microPET/CT imaging indicate its ability to penetrate brain and label parenchymal plaques in transgenic mice. Following further validation of its performance in other AD rodent models and nonhuman primates, Fluselenamyl could offer a platform technology for monitoring earliest stages of Aß pathophysiology in vivo.

Gradient-Based Algorithm for Determining Tumor Volumes in Small Animals Using Planar Fluorescence Imaging Platform.

Planar fluorescence imaging is widely used in biological research because of its simplicity, use of non-ionizing radiation, and high-throughput data acquisition. In cancer research, where small animal models are used to study the in vivo effects of cancer therapeutics, the output of interest is often the tumor volume. Unfortunately, inaccuracies in determining tumor volume from surface-weighted projection fluorescence images undermine the data, and alternative physical or conventional tomographic approaches are prone to error or are tedious for most laboratories. Here, we report a method that uses a priori knowledge of a tumor xenograft model, a tumor-targeting near infrared probe, and a custom-developed image analysis planar view tumor volume algorithm (PV-TVA) to estimate tumor volume from planar fluorescence images. Our algorithm processes images obtained using near infrared light for improving imaging depth in tissue in comparison with light in the visible spectrum. We benchmarked our results against the actual tumor volume obtained from a standard water volume displacement method. Compared with a caliper-based method that has an average deviation from an actual volume of 18% (204.34 ± 115.35 mm3), our PV-TVA average deviation from the actual volume was 9% (97.24 ± 70.45 mm3; P < .001). Using a normalization-based analysis, we found that bioluminescence imaging and PV-TVA average deviations from actual volume were 36% and 10%, respectively. The improved accuracy of tumor volume assessment from planar fluorescence images, rapid data analysis, and the ease of archiving images for subsequent retrieval and analysis potentially lend our PV-TVA method to diverse cancer imaging applications.

(67/68)Galmydar: A metalloprobe for monitoring breast cancer resistance protein (BCRP)-mediated functional transport activity.

INTRODUCTION: For stratification of chemotherapeutic choices, radiopharmaceuticals capable of imaging breast cancer resistance protein (BCRP/ABCG2)-mediated functional transport are desired. To accomplish this objective, Galmydar, a fluorescent and moderately hydrophobic Ga(III) cationic complex and its (67/68)Ga-radiolabeled counterparts were interrogated in HEK293 cells stably transfected with BCRP and their WT counterparts transfected with empty vector. Additionally, the sensitivity and specificity of (68)Ga-Galmydar to evaluate functional expression of BCRP at the blood-brain barrier (BBB) was investigated in gene-knockout mdr1a/1b(-/-) (double knockout, dKO) and mdr1a/1b(-/-)ABCG2(-/-) (triple knockout, tKO) mouse models. METHODS: For radiotracer uptake assays and live cell fluorescence imaging, either (67)Ga-Galmydar or its unlabeled counterpart was incubated in HEK293 cells transfected with BCRP (HEK293/BCRP) and their WT counterparts at 37°C under a continuous flux of CO2 (5%) in the presence or absence of Ko143, a potent BCRP antagonist, and cellular uptake was measured to assess the sensitivity of Galmydar to probe BCRP-mediated functional transport activity in cellulo. For assessing the potential of Galmydar to enable diagnostic imaging of targeted tissues in vivo, the (67)Ga-radiolabeled counterpart was incubated in either human serum albumin or human serum at 37°C and the percentage of unbound (67)Ga-Galmydar was determined. To evaluate the sensitivity of (68)Ga-Galmydar for molecular imaging of BCRP-mediated efflux activity in vivo, microPET/CT brain imaging was performed in dKO and tKO mice and their age-matched WT counterparts, 60min post-intravenous injection. RESULTS: (67)Ga-Galmydar shows uptake profiles in HEK293 cells inversely proportional to BCRP expression, and antagonist (Ko143) induced accumulation in HEK293/BCRP cells, thus indicating target sensitivity and specificity. Furthermore, employing the fluorescent characteristics of Galmydar, optical imaging in HEK293/BCRP cells shows an excellent correlation with the radiotracer cellular accumulation data. (67)Ga-Galmydar shows > 85% unbound fraction and presence of parental compound in human serum. Finally, microPET/CT imaging shows higher retention of (68)Ga-Galmydar in brains of dKO and tKO mice compared to their age-matched WT counterparts, 60min post-intravenous tail-vein injection. CONCLUSIONS: Combined data indicate that Galmydar could provide a template scaffold for development of a PET tracer for imaging BCRP-mediated functional transport activity in vivo.

Synthesis, characterization, and preclinical validation of a PET radiopharmaceutical for interrogating Aß (ß-amyloid) plaques in Alzheimer's disease.

BACKGROUND: PET radiopharmaceuticals capable of imaging ß-amyloid (Aß) plaque burden in the brain could offer highly valuable diagnostic tools for clinical studies of Alzheimer's disease. To further supplement existing armamentarium of FDA-approved agents as well as those under development, and to correlate multiphoton-imaging data reported earlier, herein, we describe preclinical validation of a PET tracer. METHODS: A novel PET radiopharmaceutical ((18)F-7B) was synthesized and characterized. To assess its affinity for Aß, binding assays with Aß1-42 fibrils, Alzheimer's disease (AD) homogenates, and autoradiography studies and their IHC correlations were performed. For assessing its overall pharmacokinetic profiles in general and its ability to cross the blood-brain barrier (BBB) in particular, biodistribution studies in normal mice were performed. Finally, for evaluating potential for (18)F-7B to serve as a targeted Aß probe, the microPET/CT imaging was performed in age-matched amyloid precursor protein/presenilin-1 (APP/PS1) mice and wild-type (WT) counterparts. RESULTS: The radiotracer (18)F-7B shows saturable binding to autopsy-confirmed AD homogenates (K d = 17.7 nM) and Aß1-42 fibrils (K d = 61 nM). Preliminary autoradiography studies show binding of (18)F-7B to cortical Aß plaques in autopsy-confirmed AD tissue sections, inhibition of that binding by unlabeled counterpart 7A-indicating specificity, and a good correlation of tracer binding with Aß immunostaining. The agent indicates high initial penetration into brains (7.23 ± 0.47%ID/g; 5 min) of normal mice, thus indicating a 5-min/120-min brain uptake clearance ratio of 4.7, a benchmark value (>4) consistent with the ability of agents to traverse the BBB to enable PET brain imaging. Additionally, (18)F-7B demonstrates the presence of parental species in human serum. Preliminary microPET/CT imaging demonstrates significantly higher retention of (18)F-7B in brains of transgenic mice compared with their WT counterparts, consistent with expected binding of the radiotracer to Aß plaques, present in APP/PS1 mice, compared with their age-matched WT counterparts lacking those Aß aggregates. CONCLUSIONS: These data offer a platform scaffold conducive to further optimization for developing new PET tracers to study Aß pathophysiology in vitro and in vivo.

[(18)F]FHBG PET/CT Imaging of CD34-TK75 Transduced Donor T Cells in Relapsed Allogeneic Stem Cell Transplant Patients: Safety and Feasibility.

Described herein is a first-in-man attempt to both genetically modify T cells with an imagable suicide gene and track these transduced donor T cells in allogeneic stem cell transplantation recipients using noninvasive positron emission tomography/computerized tomography (PET/CT) imaging. A suicide gene encoding a human CD34-Herpes Simplex Virus-1-thymidine kinase (CD34-TK75) fusion enabled enrichment of retrovirally transduced T cells (TdT), control of graft-versus-host disease and imaging of TdT migration and expansion in vivo in mice and man. Analysis confirmed that CD34-TK75-enriched TdT contained no replication competent γ-retrovirus, were sensitive to ganciclovir, and displayed characteristic retroviral insertion sites (by targeted sequencing). Affinity-purified CD34-TK75(+)-selected donor T cells (1.0-13 × 10(5))/kg were infused into eight patients who relapsed after allogeneic stem cell transplantation. Six patients also were administered 9-[4-((18)F)fluoro-3-hydroxymethyl-butyl]guanine ([(18)F]FHBG) to specifically track the genetically modified donor T cells by PET/CT at several time points after infusion. All patients were assessed for graft-versus-host disease, response to ganciclovir, circulating TdT cells (using both quantitative polymerase chain reaction and [(18)F]FHBG PET/CT imaging), TdT cell clonal expansion, and immune response to the TdT. This phase 1 trial demonstrated that genetically modified T cells and [(18)F]FHBG can be safely infused in patients with relapsed hematologic malignancies after allogeneic stem cell transplantation.

A generator-produced gallium-68 radiopharmaceutical for PET imaging of myocardial perfusion.

Lipophilic cationic technetium-99m-complexes are widely used for myocardial perfusion imaging (MPI). However, inherent uncertainties in the supply chain of molybdenum-99, the parent isotope required for manufacturing 99Mo/99mTc generators, intensifies the need for discovery of novel MPI agents incorporating alternative radionuclides. Recently, germanium/gallium (Ge/Ga) generators capable of producing high quality 68Ga, an isotope with excellent emission characteristics for clinical PET imaging, have emerged. Herein, we report a novel 68Ga-complex identified through mechanism-based cell screening that holds promise as a generator-produced radiopharmaceutical for PET MPI.

The collagen receptor discoidin domain receptor 2 stabilizes SNAIL1 to facilitate breast cancer metastasis.

Increased stromal collagen deposition in human breast tumours correlates with metastases. We show that activation of the collagen I receptor DDR2 (discoidin domain receptor 2) regulates SNAIL1 stability by stimulating ERK2 activity, in a Src-dependent manner. Activated ERK2 directly phosphorylates SNAIL1, leading to SNAIL1 nuclear accumulation, reduced ubiquitylation and increased protein half-life. DDR2-mediated stabilization of SNAIL1 promotes breast cancer cell invasion and migration in vitro, and metastasis in vivo. DDR2 expression was observed in most human invasive ductal breast carcinomas studied, and was associated with nuclear SNAIL1 and absence of E-cadherin expression. We propose that DDR2 maintains SNAIL1 level and activity in tumour cells that have undergone epithelial-mesenchymal transition (EMT), thereby facilitating continued tumour cell invasion through collagen-I-rich extracellular matrices by sustaining the EMT phenotype. As such, DDR2 could be an RTK (receptor tyrosine kinase) target for the treatment of breast cancer metastasis.

IFNγR signaling mediates alloreactive T-cell trafficking and GVHD.

The clinical goal of allogeneic hematopoietic stem cell transplantation (allo-HSCT) is to minimize GVHD while maintaining GvL. Here, we show that interferon γ receptor-deficient (IFNγR(-/-)) allogeneic Tconv, which possess normal alloreactivity and cytotoxicity, induce significantly less GVHD than wild-type (WT) Tconv. This effect is mediated by altered trafficking of IFNγR(-/-) Tconv to GVHD target organs, especially the gastrointestinal (GI) tract. We show that the chemokine receptor CXCR3 is induced via IFNγR-mediated signaling and partially contributes to the trafficking of WT Tconv to GVHD target organs. Indeed, CXCR3(-/-) Tconv recapitulate the reduced GVHD potential of IFNγR(-/-) Tconv in a minor-mismatched GVHD model. Most importantly, IFNγR(-/-) (and CXCR3(-/-)) Tconv mediate a robust and beneficial GvL effect. In addition, we show that IFNγR(-/-) regulatory T cells (Tregs) are fully suppressive in vitro although defective in suppressor function in vivo and that WT Tregs suppress GVHD in vivo only when allogeneic Tconv produce interferon γ (IFNγ), suggesting that the IFNγR signaling pathway is the major mechanism for both Tregs and Tconv to migrate to GVHD target organs. Finally, pharmacologic inhibition of IFNγR signaling with inhibitors of JAK1/JAK2, which are mediators of IFNγR signaling, results in the decreased expression of CXCR3 and reduced GVHD and improved survival after allo-HSCT and this effect is mediated by altered trafficking of Tconv to GVHD target organs.

Breast cancer cell targeting by prenylation inhibitors elucidated in living animals with a bioluminescence reporter.

PURPOSE: Inhibitors of protein prenylation, including prenyltransferase inhibitors and aminobisphosphonates such as zoledronic acid, are being investigated intensively as therapeutics in cancer and other diseases. Determining whether prenylation inhibitors directly or indirectly target tumor and/or host cells is key to understanding therapeutic mechanisms. EXPERIMENTAL DESIGN: To determine which cell types can be targeted directly by distinct classes of prenylation inhibitors in vivo, we describe herein the development and implementation of a sensitive and pharmacologically specific bioluminescence-based imaging reporter that is inducible by prenylation inhibitors. RESULTS: In mouse xenograft models of breast cancer, using reporter-bearing mammary fat pad- or bone-localized tumor cells, we show that a prenyltransferase inhibitor robustly induces reporter activity in vivo. In contrast, zoledronic acid, a bone-associated aminobisphosphonate that exerts adjuvant chemotherapeutic activity in patients with breast cancer, fails to induce reporter activity in tumor cells of either model. CONCLUSIONS: Although a prenyltransferase inhibitor can directly target breast cancer cells in vivo, zoledronic acid and related aminobisphosphonates are likely to exert antitumor activity indirectly by targeting host cells. Accordingly, these findings shift attention toward the goal of determining which host cell types are targeted directly by aminobisphosphonates to exert adjuvant chemotherapeutic activity.

Hedgehog signaling inhibition blocks growth of resistant tumors through effects on tumor microenvironment.

Hedgehog (Hh) signaling is implicated in bone development and cellular transformation. Here we show that inhibition of Hh pathway activity inhibits tumor growth through effects on the microenvironment. Pharmacologic inhibition of the Hh effector Smoothened (Smo) increased trabecular bone in vivo and inhibited osteoclastogenesis in vitro. In addition, enhanced Hh signaling due to heterozygosity of the Hh inhibitory receptor Patched (Ptch1(+/-)) increased bone resorption, suggesting direct regulation of osteoclast (OC) activity by the Hh pathway. Ptch1(+/-) mice had increased bone metastatic and subcutaneous tumor growth, suggesting that increased Hh activation in host cells promoted tumor growth. Subcutaneous growth of Hh-resistant tumor cells was inhibited by LDE225, a novel orally bioavailable SMO antagonist, consistent with effects on tumor microenvironment. Knockdown of the Hh ligand Sonic Hh (SHH) in these cells decreased subcutaneous tumor growth and decreased stromal cell production of interleukin-6, indicating that tumor-derived Hh ligands stimulated tumor growth in a paracrine fashion. Together our findings show that inhibition of the Hh pathway can reduce tumor burden, regardless of tumor Hh responsiveness, through effects on tumor cells, OCs, and stromal cells within the tumor microenvironment. Hh may be a promising therapeutic target for solid cancers and bone metastases.

Synthesis, characterization, and molecular structure of a novel zinc (II) complex: assessment of impact of MDR1Pgp expression on its cytotoxic activity.

Zinc(II)complex (3) {bis(3-ethoxy-2-hydroxy-benzylidene)-N,N'-bis(2,2-dimethyl-3-aminopropyl)ethylenediamine}-zinc(II); [(3-OEt-ENBDMPI)Zn(II)] was obtained in situ by a ligand exchange reaction involving zinc(II) acetylacetonate and the Schiff-base ligand obtained in situ. For assessing ability of 3 to act as a transport substrate of multidrug resistance (MDR1) P-glycoprotein (Pgp), its cytotoxic activity was evaluated in human epidermal carcinoma drug-sensitive KB 3-1 (Pgp-) and drug resistant KB 8-5 (Pgp+) cells. Compared with its cationic gallium(III) counterpart 4 showing cytotoxicity profiles consistent with its recognition as a Pgp substrate, the neutral zinc(II) complex 3 did not display cytotoxicity profiles (at pharmacologically relevant concentrations <10 µM) modified by expression of Pgp. Further, 3 was found be slightly more toxic against KB 8-5 cells compared to KB 3-1 cells at higher concentration. The neutral zinc (II) complex 3 was also found to be considerably less toxic against Pgp-lacking cells compared to its cationic gallium(III) counterpart 4. Additionally, the neutral zinc(II) complex 3 demonstrated considerably more toxicity against Pgp expressing KB 8-5 cells (> 10 µM) compared with its cationic counterpart 4 displaying minimal effect at highest concentration. The results suggest that differential cytotoxic activity of 3 and 4 in drug-resistant human epidermal carcinoma KB 8-5 (Pgp+) cells could result from variation in the overall charge of the molecules.

Monitoring molecular-specific pharmacodynamics of rapamycin in vivo with inducible Gal4->Fluc transgenic reporter mice.

Rapamycin (Rap), a small-molecule inhibitor of mTOR, is an immunosuppressant, and several Rap analogues are cancer chemotherapeutics. Further pharmacologic development will be significantly facilitated if in vivo reporter models are available to enable monitoring of molecular-specific pharmacodynamic actions of Rap and its analogues. Herein we present the use of a Gal4→Fluc reporter mouse for the study of Rap-induced mTOR/FKBP12 protein-protein interactions in vivo with the use of a mouse two-hybrid transactivation strategy, a derivative of the yeast two-hybrid system applied to live mice. Upon treatment with Rap, a bipartite transactivator was reconstituted, and transcription of a genomic firefly luciferase reporter was activated in a concentration-dependent (K(d) = 2.3 nmol/L) and FK506-competitive (K(i) = 17.1 nmol/L) manner in cellulo, as well as in a temporal and specific manner in vivo. In particular, after a single dose of Rap (4.5 mg/kg, i.p.), peak Rap-induced protein-protein interactions were observed in the liver at 24 hours post treatment, with photon flux signals 600-fold over baseline, which correlated temporally with suppression of p70S6 kinase activity, a downstream effector of mTOR. The Gal4→Fluc reporter mouse provides an intact physiologic system to interrogate protein-protein interactions and molecular-specific pharmacodynamics during drug discovery and lead characterization. Imaging protein interactions and functional proteomics in whole animals in vivo may serve as a basic tool for screening and mechanism-based analysis of small molecules targeting specific protein-protein interactions in human diseases.

Loss of Par-1a/MARK3/C-TAK1 kinase leads to reduced adiposity, resistance to hepatic steatosis, and defective gluconeogenesis.

Par-1 is an evolutionarily conserved protein kinase required for polarity in worms, flies, frogs, and mammals. The mammalian Par-1 family consists of four members. Knockout studies of mice implicate Par-1b/MARK2/EMK in regulating fertility, immune homeostasis, learning, and memory as well as adiposity, insulin hypersensitivity, and glucose metabolism. Here, we report phenotypes of mice null for a second family member (Par-1a/MARK3/C-TAK1) that exhibit increased energy expenditure, reduced adiposity with unaltered glucose handling, and normal insulin sensitivity. Knockout mice were protected against high-fat diet-induced obesity and displayed attenuated weight gain, complete resistance to hepatic steatosis, and improved glucose handling with decreased insulin secretion. Overnight starvation led to complete hepatic glycogen depletion, associated hypoketotic hypoglycemia, increased hepatocellular autophagy, and increased glycogen synthase levels in Par-1a(-/-) but not in control or Par-1b(-/-) mice. The intercrossing of Par-1a(-/-) with Par-1b(-/-) mice revealed that at least one of the four alleles is necessary for embryonic survival. The severity of phenotypes followed a rank order, whereby the loss of one Par-1b allele in Par-1a(-/-) mice conveyed milder phenotypes than the loss of one Par-1a allele in Par-1b(-/-) mice. Thus, although Par-1a and Par-1b can compensate for one another during embryogenesis, their individual disruption gives rise to distinct metabolic phenotypes in adult mice.

Synthesis, molecular structure, and validation of metalloprobes for assessment of MDR1 P-glycoprotein-mediated functional transport.

UNLABELLED: The human genome is known to consist of 49 ATP-binding cassette (ABC) transporter genes. Among these ABC proteins, overexpression of multidrug resistance (MDR1) P-glycoprotein (Pgp/ABCB1) is the best characterized barrier to successful chemotherapeutic treatments, impacts pharmacokinetics of numerous recognized drugs, and is also quickly emerging as an important target in the development of neurodegenerative diseases. Therefore, there exists an urgent need to seek radiopharmaceuticals, incorporated with generator-produced radionuclides to assist their widespread deployment, for noninvasive assessment of Pgp-mediated functional transport activity in vivo. METHODS: gallium(III) complexes (5a and 5b) possessing octahedral geometry were synthesized, analytically characterized, and evaluated for their potential to serve as probes of Pgp-mediated functional transport activity in cellulo and in vivo. While unlabeled compounds (5a and 5b) were examined via cell cytotoxicity assays, the (67)Ga-labeled counterparts (6a and 6b) were evaluated via cell transport studies and quantitative biodistribution studies in mdr1a/1b((-/-)) gene-deleted mice and their wild-type (WT) counterparts. RESULTS: cytotoxicity data of 5a and 5b displayed profiles modified by the expression of Pgp in drug-resistant cells. (67)Ga-metalloprobes (6a and 6b) showed high accumulation in human epidermal carcinoma drug-sensitive KB-3-1 cells (Pgp-), human breast carcinoma MCF-7 (Pgp-) cells; an inhibitor (LY335979, 1 microM) induced accumulation in multidrug resistant (MDR, Pgp+) KB-8-5, KB-8-5-11 cells, and stably transfected MCF-7/MDR1 cells, thus demonstrating their ability to interrogate Pgp-mediated functional transport activity in cellulo. In mdr1a/1b((-/-)) gene-deleted mice, the (67)Ga-metalloprobe (6b) showed 8-fold greater brain uptake and retention compared with WT counterparts and no significant difference in blood pharmacokinetics. CONCLUSION: molecular imaging of the functional transport activity of MDR1 Pgp (ABCB1) with (67/68)Ga-metalloprobes could enable non-invasive monitoring of the blood-brain barrier, tumors, and tissues in vivo.

In vivo administration of hypomethylating agents mitigate graft-versus-host disease without sacrificing graft-versus-leukemia.

Regulatory T cells (Tregs) suppress graft-versus-host disease (GVHD) while preserving a beneficial graft-versus-leukemia (GVL) effect. Thus, their use in allogeneic stem cell transplantation (SCT) provides a promising strategy to treat GVHD. However, 3 obstacles prevent their routine use in human clinical trials: (1) low circulating number of Tregs in peripheral blood, (2) loss of suppressor function after in vitro expansion, and (3) lack of Treg-specific surface markers necessary for efficient purification. FOXP3 is exclusively expressed in Tregs and forced expression in CD4(+)CD25(-) T cells can convert these non-Tregs into Tregs with functional suppressor function. Here, we show that the FDA-approved hypomethylating agents, decitabine (Dec) and azacitidine (AzaC), induce FOXP3 expression in CD4(+)CD25(-) T cells both in vitro and in vivo. Their suppressor function is dependent on direct contact, partially dependent on perforin 1 (Prf1), but independent of granzyme B (GzmB), and surprisingly, Foxp3. Independence of Foxp3 suggests that genes responsible for the suppressor function are also regulated by DNA methylation. We have identified 48 candidate genes for future studies. Finally, AzaC treatment of mice that received a transplant of major histocompatibility complex mismatched allogeneic bone marrow and T cells mitigates GVHD while preserving GVL by peripheral conversion of alloreactive effector T cells into FOXP3(+) Tregs and epigenetic modulation of genes downstream of Foxp3 required for the suppressor function of Tregs.