CTLA-4 blockade drives loss of Treg stability in glycolysis-low tumours.

Limiting metabolic competition in the tumour microenvironment may increase the effectiveness of immunotherapy. Owing to its crucial role in the glucose metabolism of activated T cells, CD28 signalling has been proposed as a metabolic biosensor of T cells1. By contrast, the engagement of CTLA-4 has been shown to downregulate T cell glycolysis1. Here we investigate the effect of CTLA-4 blockade on the metabolic fitness of intra-tumour T cells in relation to the glycolytic capacity of tumour cells. We found that CTLA-4 blockade promotes metabolic fitness and the infiltration of immune cells, especially in glycolysis-low tumours. Accordingly, treatment with anti-CTLA-4 antibodies improved the therapeutic outcomes of mice bearing glycolysis-defective tumours. Notably, tumour-specific CD8+ T cell responses correlated with phenotypic and functional destabilization of tumour-infiltrating regulatory T (Treg) cells towards IFNγ- and TNF-producing cells in glycolysis-defective tumours. By mimicking the highly and poorly glycolytic tumour microenvironments in vitro, we show that the effect of CTLA-4 blockade on the destabilization of Treg cells is dependent on Treg cell glycolysis and CD28 signalling. These findings indicate that decreasing tumour competition for glucose may facilitate the therapeutic activity of CTLA-4 blockade, thus supporting its combination with inhibitors of tumour glycolysis. Moreover, these results reveal a mechanism by which anti-CTLA-4 treatment interferes with Treg cell function in the presence of glucose.

5-ALA fluorescence-guided resection of pediatric low-grade glioma using the ORBEYE 3D digital exoscope: a technical report.

OBJECTIVE: A case of low-grade glioma in which 5-aminolevulinic acid (5-ALA) fluorescence was visualized by a digital exoscope is presented. CASE PRESENTATION: A 14-year-old girl with recurrent paroxysmal episodes of a strange smell and nausea underwent magnetic resonance imaging (MRI) for further investigation. The MRI showed a tumor with an enhanced nodule in the right temporal lobe. The patient received 5-ALA preoperatively, and intraoperative observation using a 4 K-3-dimension digital exoscope (Olympus ORBEYE) showed that the tumor was fluorescent, which was useful in determining the extent of tumor removal. Postoperative MRI showed that the tumor was completely removed. The histopathological diagnosis was pleomorphic xanthoastrocytoma. She was discharged without any complications. CONCLUSIONS: 5-ALA-fluorescence-guided resection of low-grade glioma using the ORBEYE was useful for determining the extent of removal.

Molecular dissection of Caenorhabditis elegans ATP-binding cassette transporter protein HAF-4 to investigate its subcellular localization and dimerization.

Caenorhabditis elegans HAF-4 and HAF-9 are half-type ATP-binding cassette (ABC) transporter proteins, which are highly homologous to the human peptide transporter protein, transporter associated with antigen processing-like (TAPL, ABCB9). TAPL forms homodimers and localizes to lysosomes, whereas HAF-4 and HAF-9 form heterodimers and localize to intestine-specific non-acidified organelles. Both TAPL and HAF-4/HAF-9 are predicted to have four amino-terminal transmembrane helices [transmembrane domain 0 (TMD0)] additional to the six transmembrane helices that form the canonical core domain of ABC transporters with a cytosolic ABC region. TAPL requires its amino-terminal domain for localization to lysosomes; however, molecular mechanisms underlying HAF-4 and HAF-9 localization to their target organelles had not been elucidated. Here, we demonstrate that the mechanisms underlying HAF-4 localization differ from those underlying TAPL localization. Using transgenic C. elegans expressing mutant HAF-4 proteins labeled with green fluorescent protein, we reveal that the TMD0 of HAF-4 was not sufficient for proper localization of the protein. The mutant HAF-4, which lacked TMD0, localized to intracellular organelles similarly to the wild-type protein and functioned normally in the biogenesis of its localizing organelles, indicating that the TMD0 of HAF-4 is dispensable for both its localization and function.

Anisomycin-induced GATA-6 degradation accompanying a decrease of proliferation of colorectal cancer cell.

Transcription factor GATA-6 plays a key role in normal cell differentiation of the mesoderm and endoderm. On the other hand, GATA-6 is abnormally overexpressed in many clinical gastrointestinal cancer tissue samples, and accelerates cell proliferation or an anti-apoptotic response in cancerous tissues. We previously showed that activation of the JNK signaling cascade causes proteolysis of GATA-6. In this study, we demonstrated that anisomycin, a JNK activator, stimulates nuclear export of GATA-6 in a colorectal cancer cell line, DLD-1. Concomitantly, anisomycin remarkably inhibits the proliferation of DLD-1 cells via G2/M arrest in a plate culture. However, it did not induce apoptosis under growth arrest conditions. Furthermore, the growth of DLD-1 cells in a spheroid culture was suppressed by anisomycin. Although 5-FU showed only a slight inhibitory effect on 3D spheroid cultures, the same concentration of 5-FU together with a low concentration of anisomycin exhibited strong growth inhibition. These results suggest that the induction of GATA-6 dysfunction may be more effective for chemotherapy for colorectal cancer, although the mechanism underlying the synergistic effect of 5-FU and anisomycin remains unknown.

A unique F-type H⁺-ATPase from Streptococcus mutans: an active H⁺ pump at acidic pH.

We have shown previously that the Streptococcus mutans F-type H(+)-ATPase (F(O)F(1)) c subunit gene could complement Escherichia coli defective in the corresponding gene, particularly at acidic pH (Araki et al., (2013) [14]). In this study, the entire S. mutans F(O)F(1) was functionally assembled in the E. coli plasma membrane (SF(O)F(1)). Membrane SF(O)F(1) ATPase showed optimum activity at pH 7, essentially the same as that of the S. mutans, although the activity of E. coli F(O)F(1) (EF(O)F(1)) was optimum at pH≥9. The membranes showed detectable ATP-dependent H(+)-translocation at pH 5.5-6.5, but not at neutral conditions (pH≥7), consistent with the role of S. mutans F(O)F(1) to pump H(+) out of the acidic cytoplasm. A hybrid F(O)F(1), consisting of membrane-integrated F(O) and -peripheral F(1) sectors from S. mutans and E. coli (SF(O)EF(1)), respectively, essentially showed the same pH profile as that of EF(O)F(1) ATPase. However, ATP-driven H(+)-transport was similar to that by SF(O)F(1), with activity at acidic pH. Replacement of the conserved c subunit Glu53 in SF(O)F(1) abolished H(+)-transport at pH 6 or 7, suggesting its role in H(+) transport. Mutations in the SF(O)F(1) c subunit, Ser17Ala or Glu20Ile, changed the pH dependency of H(+)-transport, and the F(O) could transport H(+) at pH 7, as the membranes with EF(O)F(1). Ser17, Glu20, and their vicinity were suggested to be involved in H(+)-transport in S. mutans at acidic pH.

Amino-terminal extension of 146 residues of L-type GATA-6 is required for transcriptional activation but not for self-association.

Transcription factor GATA-6 plays essential roles in developmental processes and tissue specific functions through regulation of gene expression. GATA-6 mRNA utilizes two Met-codons in frame as translational initiation codons. Deletion of the nucleotide sequence encoding the PEST sequence (Glu(31)-Cys(46)) between the two initiation codons unusually reduced the protein molecular size on SDS-polyacrylamide gel-electrophoresis, and re-introduction of this sequence reversed this change. The long-type (L-type) GATA-6 containing this PEST sequence self-associated similarly to the short-type (S-type) GATA-6, as determined on co-immunoprecipitation of Myc-tagged GATA-6 with HA-tagged GATA-6. The L-type and S-type GATA-6 also interacted mutually. The L-type GATA-6 without the PEST sequence also self-associated and interacted with the S-type GATA-6. The transcriptional activation potential of L-type GATA-6 is higher than that of S-type GATA-6. When the PEST sequence (Glu(31)-Cys(46)) was inserted into the L-type GATA-6 without Arg(13)-Gly(101), the resultant recombinant protein showed significantly higher transcriptional activity, while the construct with an unrelated sequence exhibited lower activity. These results suggest that the Glu(31)-Cys(46) segment plays an important role in the transcriptional activation, although it does not participate in the self-association.

Co-operative function and mutual stabilization of the half ATP-binding cassette transporters HAF-4 and HAF-9 in Caenorhabditis elegans.

Caenorhabditis elegans HAF-4 and HAF-9 are half ABC (ATP-binding-cassette) transporters that are highly homologous to the human lysosomal peptide transporter TAPL [TAP (transporter associated with antigen processing)-like; ABCB9]. We reported previously that both HAF-4 and HAF-9 localize to the membrane of a subset of intestinal organelles, and are required for the formation of these organelles and other physiological aspects. In the present paper, we report the genetic and physical interactions between HAF-4 and HAF-9. Overexpression of HAF-4 and HAF-9 did not rescue the intestinal organelle defect of the haf-9 and haf-4 deletion mutants respectively, indicating that they cannot substitute for each other. Double haf-4 and haf-9 mutants do not exhibit more severe phenotypes than the single mutants, suggesting their co-operative function. Immunoprecipitation experiments demonstrated their physical interaction. The results of the present study suggest that HAF-4 and HAF-9 form a heterodimer. Furthermore, Western blot analysis of the deletion mutants and RNAi (RNA interference) knockdown experiments in GFP (green fluorescent protein)-tagged HAF-4 or HAF-9 transgenic worms suggest that HAF-4-HAF-9 heterodimer formation is required for their stabilization. The findings provide a clue as to how ABC transporters adopt a stable functional form.

Complementation of the Fo c subunit of Escherichia coli with that of Streptococcus mutans and properties of the hybrid FoF1 ATP synthase.

The c subunit of Streptococcus mutans ATP synthase (FoF1) is functionally exchangeable with that of Escherichia coli, since E. coli with a hybrid FoF1 is able to grow on minimum succinate medium through oxidative phosphorylation. E. coli F1 bound to the hybrid Fo with the S. mutans c subunit showed N,N'-dicyclohexylcarbodiimide-sensitive ATPase activity similar to that of E. coli FoF1. Thus, the S. mutans c subunit assembled into a functional Fo together with the E. coli a and b subunits, forming a normal F1 binding site. Although the H(+) pathway should be functional, as was suggested by the growth on minimum succinate medium, ATP-driven H(+) transport could not be detected with inverted membrane vesicles in vitro. This observation is partly explained by the presence of an acidic residue (Glu-20) in the first transmembrane helix of the S. mutans c subunit, since the site-directed mutant carrying Gln-20 partly recovered the ATP-driven H(+) transport. Since S. mutans is recognized to be a primary etiological agent of human dental caries and is one cause of bacterial endocarditis, our system that expresses hybrid Fo with the S. mutans c subunit would be helpful to find antibiotics and chemicals specifically directed to S. mutans.

Cyclic AMP (cAMP)-dependent proteolysis of GATA6 by proteasome: Zinc-finger domain of GATA6 has signals for nuclear export and proteolysis, both of which are responsive to cAMP.

Transcription factor GATA6 stably expressed in Chinese hamster ovary (CHO)-K1 cells is exported from the nucleus to the cytoplasm and degraded there by proteasome upon treatment with dibutylyl-cyclic AMP (dbcAMP), which is a membrane-permeable cyclic AMP (cAMP) analogue. The cAMP-dependent proteolysis of GATA6 was characterized by dissection of the GATA6 protein into a zinc-finger domain (Zf) and the surrounding region (ΔZf). These segments were separately expressed in CHO-K1 cells stably, and followed by treatment with dbcAMP. The nuclear localized Zf was degraded by proteasome similarly to the full-length GATA6. Site-directed mutants of nuclear localizing signal (NLS) (345RKRKPK350 → AAAAPK and AAAAPA) and closely related GATA4 showed the same behavior. Although nuclear-localized ΔZf was degraded by proteasome, the cytoplasmic-located ΔZf was resistant to proteolysis in contrast to the NLS mutants. We also searched for a potential NLS and nuclear export signal (NES) with computational prediction programs and compared the results with ours. All these results suggest that the amino acid sequence(s) of the Zf of GATA6 is responsive to cAMP-dependent nuclear export and proteolysis.

cAMP-dependent proteolysis of GATA-6 is linked to JNK-signaling pathway.

A JNK inhibitor SP600125 inhibited cAMP-dependent proteolysis of GATA-6 by proteasomes around its IC50. We further examined the effects of SP600125 on the degradation of GATA-6 in detail, since an activator of JNK (anisomycin) is available. Interestingly, anisomycin immediately stimulated the export of nuclear GATA-6 into the cytoplasm, and then the cytoplasmic content of GATA-6 decreased slowly through degradation by proteasomes. Such an effect of anisomycin was inhibited by SP600125, indicating that the observed phenomenon might be linked to the JNK signaling pathway. The inhibitory effect of SP600125 could not be ascribed to the inhibition of PKA, since phosphorylation of CREB occurred in the presence of dbcAMP and SP600125. The nuclear export of GATA-6 was inhibited by leptomycin B, suggesting that CRM1-mediated export could be activated by anisomycin. Furthermore, it seems likely that the JNK activated by anisomycin may stimulate not only the nuclear export of GATA-6 through CRM1 but also the degradation of GATA-6 by cytoplasmic proteasomes. In contrast, A-kinase might activate only the latter process through JNK.

LDH-A-Modulation and the Variability of LDH Isoenzyme Profiles in Murine Gliomas: A Link with Metabolic and Growth Responses.

Three murine glioma cell lines (GL261, CT2A, and ALTS1C1) were modified to downregulate the expression of the murine LDH-A gene using shRNA, and compared to shRNA scrambled control (NC) cell lines. Differences in the expression of LDH-A and LDH-B mRNA, protein and enzymatic activity, as well as their LDH isoenzyme profiles, were observed in the six cell lines, and confirmed successful LDH-A KD. LDH-A KD (knock-down) resulted in metabolic changes in cells with a reduction in glycolysis (GlycoPER) and an increase in basal respiratory rate (mitoOCR). GL261 cells had a more limited ATP production capacity compared to CT2A and ALTS1C1 cells. An analysis of mRNA expression data indicated that: (i) GL261 LDH-A KD cells may have an improved ability to metabolize lactate into the TCA cycle; and (ii) that GL261 LDH-A KD cells can upregulate lipid metabolism/fatty acid oxidation pathways, whereas the other glioma cell lines do not have this capacity. These two observations suggest that GL261 LDH-A KD cells can develop/activate alternative metabolic pathways for enhanced survival in a nutrient-limited environment, and that specific nutrient limitations have a variable impact on tumor cell metabolism and proliferation. The phenotypic effects of LDH-A KD were compared to those in control (NC) cells and tumors. LDH-A KD prolonged the doubling time of GL261 cells in culture and prevented the formation of subcutaneous flank tumors in immune-competent C57BL/6 mice, whereas GL261 NC tumors had a prolonged growth delay in C57BL/6 mice. In nude mice, both LDH-A KD and NC GL261 tumors grew rapidly (more rapidly than GL261 NC tumors in C57BL/6 mice), demonstrating the impact of an intact immune system on GL261 tumor growth. No differences between NC and KD cell proliferation (in vitro) or tumor growth in C57BL/6 mice (doubling time) were observed for CT2A and ALTS1C1 cells and tumors, despite the small changes to their LDH isoenzyme profiles. These results suggest that GL261 glioma cells (but not CT2A and ALTS1C1 cells) are pre-programmed to have the capacity for activating different metabolic pathways with higher TCA cycle activity, and that this capacity is enhanced by LDH-A depletion. We observed that the combined impact of LDH-A depletion and the immune system had a significant impact on the growth of subcutaneous-located GL261 tumors.

Genetic and Drug Inhibition of LDH-A: Effects on Murine Gliomas.

The effects of the LDH-A depletion via shRNA knockdown on three murine glioma cell lines and corresponding intracranial (i.c.) tumors were studied and compared to pharmacologic (GNE-R-140) inhibition of the LDH enzyme complex, and to shRNA scrambled control (NC) cell lines. The effects of genetic-shRNA LDH-A knockdown and LDH drug-targeted inhibition (GNE-R-140) on tumor-cell metabolism, tumor growth, and animal survival were similar. LDH-A KD and GNE-R-140 unexpectedly increased the aggressiveness of GL261 intracranial gliomas, but not CT2A and ALTS1C1 i.c. gliomas. Furthermore, the bioenergetic profiles (ECAR and OCR) of GL261 NC and LDH-A KD cells under different nutrient limitations showed that (a) exogenous pyruvate is not a major carbon source for metabolism through the TCA cycle of native GL261 cells; and (b) the unique upregulation of LDH-B that occurs in GL261 LDH-A KD cells results in these cells being better able to: (i) metabolize lactate as a primary carbon source through the TCA cycle, (ii) be a net consumer of lactate, and (iii) showed a significant increase in the proliferation rate following the addition of 10 mM lactate to the glucose-free media (only seen in GL261 KD cells). Our study suggests that inhibition of LDH-A/glycolysis may not be a general strategy to inhibit the i.c. growth of all gliomas, since the level of LDH-A expression and its interplay with LDH-B can lead to complex metabolic interactions between tumor cells and their environment. Metabolic-inhibition treatment strategies need to be carefully assessed, since the inhibition of glycolysis (e.g., inhibition of LDH-A) may lead to the unexpected development and activation of alternative metabolic pathways (e.g., upregulation of lipid metabolism and fatty-acid oxidation pathways), resulting in enhanced tumor-cell survival in a nutrient-limited environment and leading to increased tumor aggressiveness.

Conserved GC-boxes, E-box and GATA motif are essential for GATA-4 gene expression in P19CL6 cells.

The promoter of the GATA-4 gene was analyzed in P19CL6 cells. A 124bp segment containing conserved two GC-boxes and E-box was essential for the basal promoter activity, as determined with a transient luciferase reporter gene assay. However, an extended 1312 bp reporter construct but not the 124 bp segment, when ligated to the GFP gene and stably inserted into the chromosome, showed regulated promoter activity since GFP was expressed upon DMSO addition. Mutations of the two GC-boxes and/or E-box significantly impaired the GFP expression. Furthermore, mutation of the distal conserved GATA motif in the 1312 bp sequence decreased the expression of GFP. Chromatin immuno-precipitation assay showed that GATA-6 binds to this conserved GATA motif. These results suggest that the distal GATA motif recognized by GATA-6 together with the GC- and E-boxes may be important for transcriptional activation of the GATA-4 gene in the chromosome.

Transporter associated with antigen processing-like (ABCB9) stably expressed in Chinese hamster ovary-K1 cells is sorted to the microdomains of lysosomal membranes.

The carboxyl terminus of a human ATP-binding cassette (ABC) transporter, transporter associated with antigen processing (TAP)-like (TAPL), was tagged with green fluorescence protein (GFP), and the resulting fusion protein (TAPL-GFP) was stably expressed in Chinese hamster ovary (CHO)-K1 cells. The GFP signal was co-localized with that of LysoTracker but not that of MitoTracker, as visualized under a microscope. TAPL-GFP was co-sedimented with lysosomal marker cathepsin D on Percoll density gradient centrifugation. These results indicated that TAPL is a lysosomal ABC transporter but not a mitochondrial one. It was not solubilized completely with a non-ionic detergent under ice-cold conditions, and was co-sedimented with flotillin-1 on sucrose density gradient centrifugation. A similar result was obtained with high pH-treatment. Furthermore, treatment with methyl-ß-cyclodextrin resulted in an altered distribution of TAPL-GFP. These results suggest that TAPL may be localized to the microdomains (lipid rafts) of lysosomal membranes enriched in cholesterol.

Ectopic expression of Id1 or Id3 inhibits transcription of the GATA-4 gene in P19CL6 cells under differentiation condition.

Inhibitor of DNA binding (Id) is a dominant negative form of the E-box binding basic-helix-loop-helix (bHLH) transcription factor since it is devoid of the basic region required for DNA binding and forms an inactive hetero dimer with bHLH proteins. The E-box sequence located in the promoter region of the GATA-binding protein 4 (GATA-4) gene is essential for transcriptional activation in P19CL6 cells. These cells differentiate into cardiomyocytes and start to express GATA-4, which further triggers cardiac-specific gene expression. In this study, expression plasmids for Ids tagged with human influenza hemagglutinin (HA)-FLAG were constructed and introduced into P19CL6 cells. The stable clones expressing the recombinant Id proteins (Id1 or Id3) were isolated. The GATA-4 gene expression in these clones under differentiation condition in the presence of 1% dimethyl sulfoxide (DMSO) was repressed, with concomitant abolishment of the transcription of α-myosin heavy chain (α-MHC), which is a component of cardiac myofibrils. Thus, the increased expression of Id protein could affect GATA-4 gene expression and negatively regulate the differentiation of P19CL6 cells.

Convergent synthesis of fluorescence-labeled probes of Annonaceous acetogenins and visualization of their cell distribution.

The convergent synthesis of fluorescence-labeled solamin, an antitumor Annonaceous acetogenin, was accomplished by two asymmetric alkynylations of 2,5-diformyl tetrahydrofuran with an alkyne tagged with fluorescent groups and another alkyne with an α,ß-unsaturated γ-lactone. Assay for the growth inhibitory activity against human cancer cell lines revealed that the probe with the fluorescent groups at the end of the hydrocarbon chain may have the same mode of action as natural acetogenins. The merged fluorescence of dansyl-labeled solamin and MitoTracker Red suggests that Annonaceous acetogenins localize in the mitochondria.

[H(+)-transporting ATP synthases: insights into how their electrochemically driven motor might serve as a drug target].

ATP synthases, widely distributed in bacteria, eukaryotic mitochondria and chloroplasts, are highly conserved multi-subunit complexes. Although the conserved acidic residue in the transmembrane helix of the c subunit functions in proton transport, the surrounding residues differ among species. Such divergence could lead to different regulatory modes since pH-dependent proton transport has been demonstrated in Escherichia coli with a c subunit carrying an additional acidic residue in the helix. There is further divergence in the number of c subunits that form the ring structure in F(0). Recently, it was also suggested that certain chemicals recognize the a and c subunits of pathogenic bacterial F(0). Since there may be structural divergence even in well-conserved ATP synthases, the c subunit-ring as well as the a subunit in F(0) could be targets for drugs for specific bacterial species.

Imaging CXCR4 Expression with Iodinated and Brominated Cyclam Derivatives.

PURPOSE: CXCR4 is one of several "chemokine" receptors expressed on malignant tumors (including GBM and PCNSL) and hematopoietic stem cells. Although 68Ga-pentixafor and 68Ga-NOTA-NFB have been shown to effectively image CXCR4 expression in myeloma and other systemic malignancies, imaging CXCR4 expression in brain tumors has been more limited due to the blood-brain barrier (BBB) and a considerable fraction of CXCR4 staining is intracellular. METHODS: We synthesized 6 iodinated and brominated cyclam derivatives with high affinity (low nM range) for CXCR4, since structure-based estimates of lipophilicity suggested rapid transfer across the BBB and tumor cell membranes. RESULTS: We tested 3 iodinated and 3 brominated cyclam derivatives in several CXCR4(+) and CXCR4(-) cell lines, with and without cold ligand blocking. To validate these novel radiolabeled cyclam derivatives for diagnostic CXCR4 imaging efficacy in brain tumors, we established appropriated murine models of intracranial GBM and PCNSL. Based on initial studies, 131I-HZ262 and 76Br-HZ270-1 were shown to be the most avidly accumulated radioligands. 76Br-HZ270-1 was selected for further study in the U87-CXCR4 and PCNSL #15 intracranial tumor models, because of its high uptake (9.5 ± 1.3 %ID/g, SD) and low non-specific uptake (1.6 ± 0.7 %ID/g, SD) in the s.c. U87-CXCR4 tumor models. However, imaging CXCR4 expression in intracranial U87-CXCR4 and PCNSL #15 tumors with 76Br-HZ270-1 was unsuccessful, following either i.v. or spinal-CSF injection. CONCLUSIONS: Imaging CXCR4 expression with halogenated cyclam derivatives was successful in s.c. located tumors, but not in CNS located tumors. This was largely due to the following: (i) the hydrophilicity of the radiolabeled analogues-as reflected in the "measured" radiotracer distribution (LogD) in octanol/PBS-which stands in contrast to the structure-based estimate of LogP, which was the rationale for initiating the study and (ii) the presence of a modest BTB in intracranial U87-CXCR4 gliomas and an intact BBB/BTB in the intracranial PCNSL animal model.

Targeted Brain Tumor Radiotherapy Using an Auger Emitter.

PURPOSE: Glioblastoma multiforme is a highly aggressive form of brain cancer whose location, tendency to infiltrate healthy surrounding tissue, and heterogeneity significantly limit survival, with scant progress having been made in recent decades. EXPERIMENTAL DESIGN: 123I-MAPi (Iodine-123 Meitner-Auger PARP1 inhibitor) is a precise therapeutic tool composed of a PARP1 inhibitor radiolabeled with an Auger- and gamma-emitting iodine isotope. Here, the PARP inhibitor, which binds to the DNA repair enzyme PARP1, specifically targets cancer cells, sparing healthy tissue, and carries a radioactive payload within reach of the cancer cells' DNA. RESULTS: The high relative biological efficacy of Auger electrons within their short range of action is leveraged to inflict DNA damage and cell death with high precision. The gamma ray emission of 123I-MAPi allows for the imaging of tumor progression and therapy response, and for patient dosimetry calculation. Here we demonstrated the efficacy and specificity of this small-molecule radiotheranostic in a complex preclinical model. In vitro and in vivo studies demonstrate high tumor uptake and a prolonged survival in mice treated with 123I-MAPi when compared with vehicle controls. Different methods of drug delivery were investigated to develop this technology for clinical applications, including convection enhanced delivery and intrathecal injection. CONCLUSIONS: Taken together, these results represent the first full characterization of an Auger-emitting PARP inhibitor which demonstrate a survival benefit in mouse models of GBM and confirm the high potential of 123I-MAPi for clinical translation.

Lactate Dehydrogenase A Depletion Alters MyC-CaP Tumor Metabolism, Microenvironment, and CAR T Cell Therapy.

To enhance human prostate-specific membrane antigen (hPSMA)-specific chimeric antigen receptor (CAR) T cell therapy in a hPSMA+ MyC-CaP tumor model, we studied and imaged the effect of lactate dehydrogenase A (LDH-A) depletion on the tumor microenvironment (TME) and tumor progression. Effective LDH-A short hairpin RNA (shRNA) knockdown (KD) was achieved in MyC-CaP:hPSMA+ Renilla luciferase (RLuc)-internal ribosome entry site (IRES)-GFP tumor cells, and changes in tumor cell metabolism and in the TME were monitored. LDH-A downregulation significantly inhibited cell proliferation and subcutaneous tumor growth compared to control cells and tumors. However, total tumor lactate concentration did not differ significantly between LDH-A knockdown and control tumors, reflecting the lower vascularity, blood flow, and clearance of lactate from LDH-A knockdown tumors. Comparing treatment responses of MyC-CaP tumors with LDH-A depletion and/or anti-hPSMA CAR T cells showed that the dominant effect on tumor growth was LDH-A depletion. With anti-hPSMA CAR T cell treatment, tumor growth was significantly slower when combined with tumor LDH-A depletion and compared to control tumor growth (p < 0.0001). The lack of a complete tumor response in our animal model can be explained in part by (1) the lower activity of human CAR T cells against hPSMA-expressing murine tumors in a murine host, and (2) a loss of hPSMA antigen from the tumor cell surface in progressive generations of tumor cells.