Activating STING1-dependent immune signaling in TP53 mutant and wild-type acute myeloid leukemia.

DNA methyltransferase inhibitors (DNMTis) reexpress hypermethylated genes in cancers and leukemias and also activate endogenous retroviruses (ERVs), leading to interferon (IFN) signaling, in a process known as viral mimicry. In the present study we show that in the subset of acute myeloid leukemias (AMLs) with mutations in TP53, associated with poor prognosis, DNMTis, important drugs for treatment of AML, enable expression of ERVs and IFN and inflammasome signaling in a STING-dependent manner. We previously reported that in solid tumors poly ADP ribose polymerase inhibitors (PARPis) combined with DNMTis to induce an IFN/inflammasome response that is dependent on STING1 and is mechanistically linked to generation of a homologous recombination defect (HRD). We now show that STING1 activity is actually increased in TP53 mutant compared with wild-type (WT) TP53 AML. Moreover, in TP53 mutant AML, STING1-dependent IFN/inflammatory signaling is increased by DNMTi treatment, whereas in AMLs with WT TP53, DNMTis alone have no effect. While combining DNMTis with PARPis increases IFN/inflammatory gene expression in WT TP53 AML cells, signaling induced in TP53 mutant AML is still several-fold higher. Notably, induction of HRD in both TP53 mutant and WT AMLs follows the pattern of STING1-dependent IFN and inflammatory signaling that we have observed with drug treatments. These findings increase our understanding of the mechanisms that underlie DNMTi + PARPi treatment, and also DNMTi combinations with immune therapies, suggesting a personalized approach that statifies by TP53 status, for use of such therapies, including potential immune activation of STING1 in AML and other cancers.

Pharmacologic induction of innate immune signaling directly drives homologous recombination deficiency.

Poly(ADP ribose) polymerase inhibitors (PARPi) have efficacy in triple negative breast (TNBC) and ovarian cancers (OCs) harboring BRCA mutations, generating homologous recombination deficiencies (HRDs). DNA methyltransferase inhibitors (DNMTi) increase PARP trapping and reprogram the DNA damage response to generate HRD, sensitizing BRCA-proficient cancers to PARPi. We now define the mechanisms through which HRD is induced in BRCA-proficient TNBC and OC. DNMTi in combination with PARPi up-regulate broad innate immune and inflammasome-like signaling events, driven in part by stimulator of interferon genes (STING), to unexpectedly directly generate HRD. This inverse relationship between inflammation and DNA repair is critical, not only for the induced phenotype, but also appears as a widespread occurrence in The Cancer Genome Atlas datasets and cancer subtypes. These discerned interactions between inflammation signaling and DNA repair mechanisms now elucidate how epigenetic therapy enhances PARPi efficacy in the setting of BRCA-proficient cancer. This paradigm will be tested in a phase I/II TNBC clinical trial.

18F-Fluoromisonidazole Quantification of Hypoxia in Human Cancer Patients Using Image-Derived Blood Surrogate Tissue Reference Regions.

UNLABELLED: (18)F-fluoromisonidazole ((18)F-FMISO) is the most widely used PET agent for imaging hypoxia, a condition associated with resistance to tumor therapy. (18)F-FMISO equilibrates in normoxic tissues but is retained under hypoxic conditions because of reduction and binding to macromolecules. A simple tissue-to-blood (TB) ratio is suitable for quantifying hypoxia. A TB ratio threshold of 1.2 or greater is useful in discriminating the hypoxic volume (HV) of tissue; TBmax is the maximum intensity of the hypoxic region and does not invoke a threshold. Because elimination of blood sampling would simplify clinical use, we tested the validity of using imaging regions as a surrogate for blood sampling. METHODS: Patients underwent 20-min (18)F-FMISO scanning during the 90- to 140-min interval after injection with venous blood sampling. Two hundred twenty-three (18)F-FMISO patient studies had detectable surrogate blood regions in the field of view. Quantitative parameters of hypoxia (TBmax, HV) derived from blood samples were compared with values using surrogate blood regions derived from the heart, aorta, or cerebellum. In a subset of brain cancer patients, parameters from blood samples and from the cerebellum were compared for their ability to independently predict outcome. RESULTS: Vascular regions of heart showed the highest correlation to measured blood activity (R(2) = 0.84). For brain studies, cerebellar activity was similarly correlated to blood samples. In brain cancer patients, Kaplan-Meier analysis showed that image-derived reference regions had predictive power nearly identical to parameters derived from blood, thus obviating the need for venous sampling in these patients. CONCLUSION: Simple static analysis of (18)F-FMISO PET captures both the intensity (TBmax) and the spatial extent (HV) of tumor hypoxia. An image-derived region to assess blood activity can be used as a surrogate for blood sampling in quantification of hypoxia.

The effects of 5-fluoruracil treatment on 3'-fluoro-3'-deoxythymidine (FLT) transport and metabolism in proliferating and non-proliferating cultures of human tumor cells.

UNLABELLED: 3'-Fluoro-3'-deoxythymidine (FLT) positron emission tomography (PET) has been proposed for imaging thymidylate synthase (TS) inhibition. Agents that target TS and shut down de novo synthesis of thymidine monophosphate increase the uptake and retention of FLT in vitro and in vivo because of a compensating increase in the salvage pathway. Increases in both thymidine kinase-1 (TK1) and the equilibrative nucleoside transporter hENT1 have been reported to underlie this effect. We examined whether the effects of one TS inhibitor, 5-fluorouracil (5FU), on FLT uptake require proliferating cells and whether the effects are limited to increasing TK1 activity. METHODS: The effects of 5FU on FLT transport and metabolism, TK1 activity, and cell cycle progression were evaluated in the human tumor cell line, A549, maintained as either a proliferating or non-proliferating culture. RESULTS: There were dose-dependent increases in FLT uptake that peaked after a 10 µM 5FU exposure and then declined to baseline levels or below at higher doses in both proliferating and non-proliferating cultures. The dose-dependence for FLT uptake was mirrored by changes in TK1 activity. S phase fraction did not correlate with FLT uptake in proliferating cultures. Chemical inhibition of hENT1 reduced overall levels of FLT uptake but did not affect the low dose increase in FLT uptake. CONCLUSIONS: 5FU only affects FLT uptake in proliferating A549 cells and increases in FLT uptake are directly related to increased TK1 activity. Our studies did not support a role for hENT1 in the increased uptake of FLT after exposure to 5FU. Our studies with A549 cells support the suggestion that FLT-PET could provide a measure of TS inhibition in vivo.

The role of nucleoside/nucleotide transport and metabolism in the uptake and retention of 3'-fluoro-3'-deoxythymidine in human B-lymphoblast cells.

INTRODUCTION: Recent studies in the human adenocarcinoma cell line A549 have identified cell growth-dependent equilibrative nucleoside transporter-1 (hENT1) as a modifier of 3'-fluoro-3'-deoxythymidine (FLT) uptake and retention. In the present study, we used the ability to isolate human lymphoblastoid clones deficient in thymidine kinase 1 (TK1) to study how metabolism and nucleoside transport influence FLT uptake and retention. METHODS: Transport and metabolism of FLT were measured in the human lymphoblastoid cell line TK6 and in eight clones isolated from TK6. Four clones were TK1-proficient, while four were TK1-deficient. Both influx and efflux of FLT were measured under conditions where concentrative and equilibrative transport could be distinguished. RESULTS: Sodium-dependent concentrative FLT transport dominated over equilibrative transport mechanisms and while inhibition of hENT1 reduced FLT uptake, there were no correlations between clonal variations in hENT1 levels and FLT uptake. There was an absolute requirement of TK1 for concentration of FLT in TK6 cells. FLT uptake reached a peak after 60 min of incubation with FLT after which intracellular levels of FLT and FLT metabolites declined. Efflux was rapid and was associated with reductions in FLT and each of its metabolites. Both FLT and FLT-monophosphate were found in the efflux buffer. CONCLUSIONS: Initial rates of FLT uptake were a function of both concentrative and equilibrative transporters. TK1 activity was an absolute requirement for the accumulation of FLT. Retention was dependent on nucleoside/nucleotide efflux and retrograde metabolism of FLT nucleotides.