Targeting DNA polymerase ß elicits synthetic lethality with mismatch repair deficiency in acute lymphoblastic leukemia.

Mismatch repair (MMR) deficiency has been linked to thiopurine resistance and hypermutation in relapsed acute lymphoblastic leukemia (ALL). However, the repair mechanism of thiopurine-induced DNA damage in the absence of MMR remains unclear. Here, we provide evidence that DNA polymerase ß (POLB) of base excision repair (BER) pathway plays a critical role in the survival and thiopurine resistance of MMR-deficient ALL cells. In these aggressive resistant ALL cells, POLB depletion and its inhibitor oleanolic acid (OA) treatment result in synthetic lethality with MMR deficiency through increased cellular apurinic/apyrimidinic (AP) sites, DNA strand breaks and apoptosis. POLB depletion increases thiopurine sensitivities of resistant cells, and OA synergizes with thiopurine to kill these cells in ALL cell lines, patient-derived xenograft (PDX) cells and xenograft mouse models. Our findings suggest BER and POLB's roles in the process of repairing thiopurine-induced DNA damage in MMR-deficient ALL cells, and implicate their potentials as therapeutic targets against aggressive ALL progression.

PRPS2 mutations drive acute lymphoblastic leukemia relapse through influencing PRPS1/2 hexamer stability.

Tumor relapse is the major cause of treatment failure in childhood acute lymphoblastic leukemia (ALL), yet the underlying mechanisms are still elusive. Here, we demonstrate that phosphoribosyl pyrophosphate synthetase 2 (PRPS2) mutations drive ALL relapse through influencing PRPS1/2 hexamer stability. Ultra-deep sequencing was performed to identify PRPS2 mutations in ALL samples. The effects of PRPS2 mutations on cell survival, cell apoptosis, and drug resistance were evaluated. In vitro PRPS2 enzyme activity and ADP/GDP feedback inhibition of PRPS enzyme activity were assessed. Purine metabolites were analyzed by ultra-performance liquid-chromatography tandem mass spectrometry (UPLC-MS/MS). Integrating sequencing data with clinical information, we identified PRPS2 mutations only in relapsed childhood ALL with thiopurine therapy. Functional PRPS2 mutations mediated purine metabolism specifically on thiopurine treatment by influencing PRPS1/2 hexamer stability, leading to reduced nucleotide feedback inhibition of PRPS activity and enhanced thiopurine resistance. The 3-amino acid V103-G104-E105, the key difference between PRPS1 and PRPS2, insertion in PRPS2 caused severe steric clash to the interface of PRPS hexamer, leading to its low enzyme activity. In addition, we demonstrated that PRPS2 P173R increased thiopurine resistance in xenograft models. Our work describes a novel mechanism by which PRPS2 mutants drive childhood ALL relapse and highlights PRPS2 mutations as biomarkers for relapsed childhood ALL.

Up-regulation of ABCG1 is associated with methotrexate resistance in acute lymphoblastic leukemia cells.

Acute lymphoblastic leukemia (ALL) is a prevalent hematologic malignancy in children, and methotrexate (MTX) is a widely employed curative treatment. Despite its common use, clinical resistance to MTX is frequently encountered. In this study, an MTX-resistant cell line (Reh-MTXR) was established through a stepwise selection process from the ALL cell line Reh. Comparative analysis revealed that Reh-MTXR cells exhibited resistance to MTX in contrast to the parental Reh cells. RNA-seq analysis identified an upregulation of ATP-binding cassette transporter G1 (ABCG1) in Reh-MTXR cells. Knockdown of ABCG1 in Reh-MTXR cells reversed the MTX-resistant phenotype, while overexpression of ABCG1 in Reh cells conferred resistance to MTX. Mechanistically, the heightened expression of ABCG1 accelerated MTX efflux, leading to a reduced accumulation of MTX polyglutamated metabolites. Notably, the ABCG1 inhibitor benzamil effectively sensitized Reh-MTXR cells to MTX treatment. Moreover, the observed upregulation of ABCG1 in Reh-MTXR cells was not induced by alterations in DNA methylation or histone acetylation. This study provides insight into the mechanistic basis of MTX resistance in ALL and also suggests a potential therapeutic approach for MTX-resistant ALL in the future.

The KRAS-G12D mutation induces metabolic vulnerability in B-cell acute lymphoblastic leukemia.

Mutations in RAS pathway genes are highly prevalent in acute lymphoblastic leukemia (ALL). However, the effects of RAS mutations on ALL cell growth have not been experimentally characterized, and effective RAS-targeting therapies are being sought after. Here, we found that Reh ALL cells bearing the KRAS-G12D mutation showed increased proliferation rates in vitro but displayed severely compromised growth in mice. Exploring this divergence, proliferation assays with multiple ALL cell lines revealed that the KRAS-G12D rewired methionine and arginine metabolism. Isotope tracing results showed that KRAS-G12D promotes catabolism of methionine and arginine to support anabolism of polyamines and proline, respectively. Chemical inhibition of polyamine biosynthesis selectively killed KRAS-G12D B-ALL cells. Finally, chemically inhibiting AKT/mTOR signaling abrogated the altered amino acid metabolism and strongly promoted the in vivo growth of KRAS-G12D cells in B-ALL xenograft. Our study thus illustrates how hyperactivated AKT/mTOR signaling exerts distinct impacts on hematological malignancies vs. solid tumors.

A γ-glutamyl hydrolase lacking the signal peptide confers susceptibility to folates/antifolates in acute lymphoblastic leukemia cells.

A key cofactor of several enzymes implicated in DNA synthesis, repair, and methylation, folate has been shown to be required for normal cell growth and replication and is the basis for cancer chemotherapy using antifolates. γ-Glutamyl hydrolase (GGH) catalyzes the removal of γ-polyglutamate tails of folylpoly-/antifolylpoly-γ-glutamates to facilitate their export out of the cell, thereby maintaining metabolic homeostasis of folates or pharmacological efficacy of antifolates. However, the factors that control or modulate GGH function are not well understood. In this study, we show that intact GGH is not indispensable for the chemosensitivity and growth of acute lymphoblastic leukemia (ALL) cells, whereas GGH lacking N-terminal signal peptide (GGH-ΔN ) confers the significant drug resistance of ALL cells to the antifolates MTX and RTX. In addition, ALL cells harboring GGH-ΔN show high susceptibility to the change in folates, and glycosylation is not responsible for these phenotypes elicited by GGH-ΔN . Mechanistically, the loss of signal peptide enhances intracellular retention of GGH and its lysosomal disposition. Our findings clearly define the in vivo role of GGH in ALL cells and indicate a novel modulation of the GGH function, suggesting new avenues for ALL treatment in future.

Chemotherapy and mismatch repair deficiency cooperate to fuel TP53 mutagenesis and ALL relapse.

Chemotherapy is a standard treatment for pediatric acute lymphoblastic leukemia (ALL), which sometimes relapses with chemoresistant features. However, whether acquired drug-resistance mutations in relapsed ALL pre-exist or are induced by treatment remains unknown. Here we provide direct evidence of a specific mechanism by which chemotherapy induces drug-resistance-associated mutations leading to relapse. Using genomic and functional analysis of relapsed ALL we show that thiopurine treatment in mismatch repair (MMR)-deficient leukemias induces hotspot TP53 R248Q mutations through a specific mutational signature (thio-dMMR). Clonal evolution analysis reveals sequential MMR inactivation followed by TP53 mutation in some patients with ALL. Acquired TP53 R248Q mutations are associated with on-treatment relapse, poor treatment response and resistance to multiple chemotherapeutic agents, which could be reversed by pharmacological p53 reactivation. Our findings indicate that TP53 R248Q in relapsed ALL originates through synergistic mutagenesis from thiopurine treatment and MMR deficiency and suggest strategies to prevent or treat TP53-mutant relapse.

Bromodomains and Extra-Terminal (BET) Inhibitor JQ1 Suppresses Proliferation of Acute Lymphocytic Leukemia by Inhibiting c-Myc-Mediated Glycolysis.

BACKGROUND Acute lymphocytic leukemia (ALL) is a common blood cancer which induces high mortality in children. Bromodomains and extra-terminal (BET) protein inhibitors, such as JQ1 and ARV-825, are promising cancer therapeutic agents that can be used by targeting c-Myc. A recent work reported that JQ1 effectively attenuates ALL in vitro by suppressing cell proliferation and accelerating apoptosis. The purpose of this research was to probe into the potential mechanism of how JQ1 inhibits ALL cell proliferation in vitro. MATERIAL AND METHODS Cell viability of ALL cells were measured by CTG after treatment by JQ1. Cell cycle analysis was done by EdU and PI staining. Cell apoptosis was assessed by Annexin V/PI staining. Glycolysis was detected using Seahorse and LC-MS kits. The expression of glycolytic rate-limiting enzymes was assessed by RNA-seq, qRT-PCR, and Western blot. RESULTS JQ1 suppressed cell proliferation by arresting the cell cycle and inducing the apoptosis of acute lymphocytic leukemia cells. JQ1 inhibited cell proliferation of B-ALL cells by restraining glycolysis. Conversely, the cell cycle block of B-ALL cells induced by JQ1 was partially abolished after pretreatment with 2-Deoxy-D-glucose (2-DG), an inhibitor of glycolysis. Furthermore, JQ1 restrained the glycolysis of B-ALL cell lines by remarkably downregulating the rate-limiting enzymes of glycolysis, such as hexokinase 2, phosphofructokinase, and lactate dehydrogenase A. Moreover, the cell cycle arrest was reversed in B-ALL cells with overexpressed c-Myc treated by JQ1, which is involved in the enhancement of glycolysis. CONCLUSIONS The BET inhibitor JQ1 suppresses the proliferation of ALL by inhibiting c-Myc-mediated glycolysis, thus providing a new strategy for the treatment of ALL.

Therapy-induced mutations drive the genomic landscape of relapsed acute lymphoblastic leukemia.

To study the mechanisms of relapse in acute lymphoblastic leukemia (ALL), we performed whole-genome sequencing of 103 diagnosis-relapse-germline trios and ultra-deep sequencing of 208 serial samples in 16 patients. Relapse-specific somatic alterations were enriched in 12 genes (NR3C1, NR3C2, TP53, NT5C2, FPGS, CREBBP, MSH2, MSH6, PMS2, WHSC1, PRPS1, and PRPS2) involved in drug response. Their prevalence was 17% in very early relapse (<9 months from diagnosis), 65% in early relapse (9-36 months), and 32% in late relapse (>36 months) groups. Convergent evolution, in which multiple subclones harbor mutations in the same drug resistance gene, was observed in 6 relapses and confirmed by single-cell sequencing in 1 case. Mathematical modeling and mutational signature analysis indicated that early relapse resistance acquisition was frequently a 2-step process in which a persistent clone survived initial therapy and later acquired bona fide resistance mutations during therapy. In contrast, very early relapses arose from preexisting resistant clone(s). Two novel relapse-specific mutational signatures, one of which was caused by thiopurine treatment based on in vitro drug exposure experiments, were identified in early and late relapses but were absent from 2540 pan-cancer diagnosis samples and 129 non-ALL relapses. The novel signatures were detected in 27% of relapsed ALLs and were responsible for 46% of acquired resistance mutations in NT5C2, PRPS1, NR3C1, and TP53. These results suggest that chemotherapy-induced drug resistance mutations facilitate a subset of pediatric ALL relapses.

Application of slightly acidic electrolyzed water and ultraviolet light for Salmonella enteritidis decontamination of cell suspensions and surfaces of artificially inoculated plastic poultry transport coops and other facility surfaces.

The efficiency of combination treatment of slightly acidic electrolyzed water (SAEW) and ultraviolet light (UV) for inactivation of Salmonella enteritidis (S. enteritidis) on the surface of plastic poultry coops and other facility surfaces was evaluated in the presence of organic matter. The bactericidal activities of SAEW, UV + SAEW, and composite phenol (CP) for inactivating S. enteritidis were also compared. Moreover, a model of UV + SAEW treatment of plastic transport coops with different times and available chlorine concentrations (ACC) was developed using multiple linear regression analysis. There are differences between SAEW and CP inactivation of S. enteritidis on coops, stainless steel, and glass surfaces (P < 0.05), and there are no differences between SAEW and CP on tire surfaces (P > 0.05). Disinfection of some rough material surfaces with SAEW treatment alone under feces interference on poultry farms may need a longer treatment time and/or a higher ACC than smooth surfaces. The combined treatment of UV and SAEW showed higher inactivation efficiency of S. enteritidis compared to CP and SAEW treatment alone (P < 0.05) in pure cultures or on the facility surfaces. A complete 100% inactivation of S. enteritidis on plastic poultry coop surfaces was obtained by using UV + SAEW with an ACC of 90 mg/L for more than 70 s. The established model had a good fit that was quantified by the determination coefficient R2 (0.93) and a lack of fit test (P > 0.05). The bactericidal efficiency of UV + SAEW increased with greater ACC and increasing time. The findings of this study indicate that the combination treatment of UV and SAEW may be a promising disinfection method and could be used instead of SAEW alone, especially on rough materials in the presence of organic matter on poultry farms.

Blocking ATM-dependent NF-κB pathway overcomes niche protection and improves chemotherapy response in acute lymphoblastic leukemia.

Bone marrow (BM) niche responds to chemotherapy-induced cytokines secreted from acute lymphoblastic leukemia (ALL) cells and protects the residual cells from chemotherapeutics in vivo. However, the underlying molecular mechanisms for the induction of cytokines by chemotherapy remain unknown. Here, we found that chemotherapeutic drugs (e.g., Ara-C, DNR, 6-MP) induced the expression of niche-protecting cytokines (GDF15, CCL3 and CCL4) in both ALL cell lines and primary cells in vitro. The ATM and NF-κB pathways were activated after chemotherapy treatment, and the pharmacological or genetic inhibition of these pathways significantly reversed the cytokine upregulation. Besides, chemotherapy-induced NF-κB activation was dependent on ATM-TRAF6 signaling, and NF-κB transcription factor p65 directly regulated the cytokines expression. Furthermore, we found that both pharmacological and genetic perturbation of ATM and p65 significantly decreased the residual ALL cells after Ara-C treatment in ALL xenograft mouse models. Together, these results demonstrated that ATM-dependent NF-κB activation mediated the cytokines induction by chemotherapy and ALL resistance to chemotherapeutics. Inhibition of ATM-dependent NF-κB pathway can sensitize ALL to chemotherapeutics, providing a new strategy to eradicate residual chemo-resistant ALL cells.

AICAr suppresses cell proliferation by inducing NTP and dNTP pool imbalances in acute lymphoblastic leukemia cells.

It has been shown that 5-amino-4-imidazolecarboxamide riboside (AICAr) can inhibit cell proliferation and induce apoptosis in childhood acute lymphoblastic leukemia (ALL) cells. Although AICAr could regulate cellular energy metabolism by activating AMPK, the cytotoxic mechanisms of AICAr are still unclear. Here, we knocked out TP53 or PRKAA1 gene (encoding AMPKα1) in NALM-6 and Reh cells by using the clustered regularly interspaced short palindromic repeats/Cas9 system and found that AICAr-induced proliferation inhibition was independent of AMPK activation but dependent on p53. Liquid chromatography-mass spectrometry analysis of nucleotide metabolites indicated that AICAr caused an increase in adenosine triphosphate, deoxyadenosine triphosphate, and deoxyguanosine triphosphate levels by up-regulating purine biosynthesis, while AICAr led to a decrease in cytidine triphosphate, uridine triphosphate, deoxycytidine triphosphate, and deoxythymidine triphosphate levels because of reduced phosphoribosyl pyrophosphate production, which consequently impaired the pyrimidine biosynthesis. Ribonucleoside triphosphate (NTP) pool imbalances suppressed the rRNA transcription efficiency. Furthermore, deoxy-ribonucleoside triphosphate (dNTP) pool imbalances induced DNA replication stress and DNA double-strand breaks, followed by cell cycle arrest and apoptosis in ALL cells. Exogenous uridine could rebalance the NTP and dNTP pools by supplementing pyrimidine and then attenuate AICAr-induced cytotoxicity. Our data indicate that RNA transcription inhibition and DNA replication stress induced by NTP and dNTP pool imbalances might play a key role in AICAr-mediated cytotoxic effects on ALL cells, suggesting a potential clinical application of AICAr in future ALL therapy.-Du, L., Yang, F., Fang, H., Sun, H., Chen, Y., Xu, Y., Li, H., Zheng, L., Zhou, B.-B. S. AICAr suppresses cell proliferation by inducing NTP and dNTP pool imbalances in acute lymphoblastic leukemia cells.

Increase of PRPP enhances chemosensitivity of PRPS1 mutant acute lymphoblastic leukemia cells to 5-Fluorouracil.

Relapse-specific mutations in phosphoribosyl pyrophosphate synthetase 1 (PRPS1), a rate-limiting purine biosynthesis enzyme, confer significant drug resistances to combination chemotherapy in acute lymphoblastic leukemia (ALL). It is of particular interest to identify drugs to overcome these resistances. In this study, we found that PRPS1 mutant ALL cells specifically showed more chemosensitivity to 5-Fluorouracil (5-FU) than control cells, attributed to increased apoptosis of PRPS1 mutant cells by 5-FU. Mechanistically, PRPS1 mutants increase the level of intracellular phosphoribosyl pyrophosphate (PRPP), which causes the apt conversion of 5-FU to FUMP and FUTP in Reh cells, to promote 5-FU-induced DNA damage and apoptosis. Our study not only provides mechanistic rationale for re-targeting drug resistant cells in ALL, but also implicates that ALL patients who harbor relapse-specific mutations of PRPS1 might benefit from 5-FU-based chemotherapy in clinical settings.

Chemotherapy-induced niche perturbs hematopoietic reconstitution in B-cell acute lymphoblastic leukemia.

BACKGROUND: Considerable efforts have been devoted toward the uncovering of the molecular mechanisms underlying the maintenance of hematopoietic stem cells (HSCs) by the normal bone marrow (BM) niche. Previously, we demonstrated that a chemotherapy-induced niche, which is mainly composed of mesenchymal stem cells (MSCs), protects the residual B-cell acute lymphoblastic leukemia (B-ALL) cells from the insult of chemotherapeutic drugs. However, the roles of chemotherapy-induced niche on HSCs functions in B-ALL remain unclear. METHODS: We established an oncogenic N-MYC-driven B-ALL mouse model, which were subsequently treated with common chemotherapy drug cytarabine (Ara-C) and daunorubicin (DNR). After treatment, the structures of the BM niche were imaged by immunofluorescence staining. Then, the self-renewal and differentiation capability of the MSCs in the BM after Ara-C and DNR treatment were studied by ex vivo culture and gene expression analysis with RNA-seq and qRT-PCR. The effects of chemotherapy-induced niche on the hematopoietic reconstitution of HSCs were determined with series transplantation assay. Furthermore, the cell cycle, ROS level, mitochondrial membrane potential and cell apoptosis of HSCs were detected by flow cytometry. RESULTS: The MSCs, which is the main component of chemotherapy-induced BM niche, have decreased self-renewal capability and are prone to differentiate into adipocytes and chondrocytes. The results of gene expression analysis with RNA-seq showed that the MSCs have reduced levels of cytokines, including SCF, CXCL12, ANGPT1, VCAM1, and IL7. Furthermore, the chemotherapy-induced niche perturbed the hematopoietic reconstitution of HSCs in our N-MYC-driven B-ALL mouse model by promoting HSCs to enter cell cycle and increasing intracellular ROS levels and mitochondrial membrane potential of HSCs, which lead to the cell apoptosis of HSCs. CONCLUSIONS: Chemotherapy-induced BM niche perturbs the hematopoietic reconstitution of HSCs by increasing intracellular ROS level and inducing cell apoptosis.

HPRT1 activity loss is associated with resistance to thiopurine in ALL.

Acute lymphoblastic leukemia (ALL) is an aggressive hematological tumor resulting from the malignant transformation of lymphoid progenitors. Thiopurine is a widely used drug in the maintaining treatment of ALL. After a period of chemotherapy, 20% of pediatric patients and over 50% of adult patients will relapse. To investigate the mechanisms of drug resistance in vitro, we established the thiopurine resistant cell lines Reh-6MPR (6-MP Resistant cell) and Reh-6TGR (6-TG Resistant cell) by stepwise selection of the ALL cell line Reh. Cell viability assay revealed that 6MPR and 6TGR cells were almost 1000-fold more resistant to thiopurine comparing with the control Reh cells, and thiopurine conversion was significantly impaired in the resistant cells. Mechanistically, a same novel hypoxanthine phosphoribosyl transferase 1 (HPRT1) mutation c.495_496insA (p.V165fs) was found by whole exome sequencing in both resistant cells. The HPRT1 mutation dramaticly decreased the production of [13C5,15N4]-IMP from [13C5,15N4]-hypoxanthine (HX), showed a loss-of-funciton mechanism. Notably, re-expression the wildtype HPRT1 in Reh-6MPR cell can reverse the drug resistance and thiopurine conversion in Reh-6MPR cells. These results highlight the importance of HPRT1's activity in thiopurine resistance.

Reirradiation + hyperthermia for recurrent breast cancer en cuirasse.

BACKGROUND AND PURPOSE: Patients with irresectable locoregional recurrent breast cancer en cuirasse (BCEC) do not have effective curative treatment options. Hyperthermia, the elevation of tumor temperature to 40-45 °C, is a well-established radio- and chemotherapy sensitizer. A total of 196 patients were treated with reirradiation and hyperthermia (reRT+HT) at two Dutch institutes from 1982-2005. The palliative effect was evaluated in terms of clinical outcome and toxicity. PATIENTS AND METHODS: All patients received previous irradiation to a median dose of 50 Gy. In all, 75% of patients received 1-6 treatment modalities for previous tumor recurrences. ReRT consisted of 8 × 4 Gy given twice a week or 12 × 3 Gy given four times a week. Superficial hyperthermia was added once or twice a week. Tumor area comprised ≥½ of the ipsilateral chest wall. RESULTS: Overall clinical response rate was 72% (complete response [CR] 30%, partial response [PR] 42%, stable disease [SD] 22%, progressive disease [PD] 6%). The local progression-free rate at 1 year was 24%. Median survival was 6.9 months. Forty-three percent of our patients with CR, PR, SD after treatment remained infield progression-free until death or last follow-up. Acute ≥grade 3 toxicity occurred in 33% of patients, while late ≥grade 3 toxicity was recorded in 14% of patients. Tumor ulceration prior to treatment had a negative impact on both clinical outcome and toxicity. CONCLUSION: ReRT+HT provides sustainable palliative tumor control, despite refractory, extensive tumor growth. Compared to currently available systemic treatment options, reRT+HT is more effective with less toxicity.

Dupuytren's disease susceptibility gene, EPDR1, is involved in myofibroblast contractility.

BACKGROUND: Dupuytren's Disease is a common disorder of the connective tissue characterized by progressive and irreversible fibroblastic proliferation affecting the palmar fascia. Progressive flexion deformity appears over several months or years and although usually painless, it can result in a serious handicap causing loss of manual dexterity. There is no cure for the disease and the etiology is largely unknown. A genome-wide association study of Dupuytren's Disease identified nine susceptibility loci with the strongest genetic signal located in an intron of EPDR1, the gene encoding the Ependymin Related 1 protein. OBJECTIVE: Here, we investigate the role of EPDR1 in Dupuytren's Disease. METHODS: We research the role of EPDR1 by assessing gene expression in patient tissue and by gene silencing in fibroblast-populated collagen lattice (FPCL) assay, which is used as an in vitro model of Dupuytren's contractures. RESULTS: The three alternative transcripts produced by the EPDR1 gene are all detected in affected Dupuytren's tissue and control unaffected palmar fascia tissue. Dupuytren's tissue also contracts more in the FPCL paradigm. Dicer-substrate RNA-mediated knockdown of EPDR1 results in moderate late stage attenuation of contraction rate in FPCL, implying a role in matrix contraction. CONCLUSION: Our results suggest functional involvement of EPDR1 in the etiology of Dupuytren's Disease.

Mineralocorticoid and angiotensin II type 1 receptors in the subfornical organ mediate angiotensin II - induced hypothalamic reactive oxygen species and hypertension.

Activation of angiotensinergic pathways by central aldosterone (Aldo)-mineralocorticoid receptor (MR) pathway plays a critical role in angiotensin II (Ang II)-induced hypertension. The subfornical organ (SFO) contains both MR and angiotensin II type 1 receptors (AT1R) and can relay the signals of circulating Ang II to downstream nuclei such as the paraventricular nucleus (PVN), supraoptic nucleus (SON) and rostral ventrolateral medulla (RVLM). In Wistar rats, subcutaneous (sc) infusion of Ang II at 500ng/min/kg for 1 or 2weeks increased reactive oxygen species (ROS) as measured by dihydroethidium (DHE) staining in a nucleus - specific pattern. Intra-SFO infusion of AAV-MR- or AT1aR-siRNA prevented the Ang II-induced increase in AT1R mRNA expression in the SFO and decreased MR mRNA. Both MR- and AT1aR-siRNA prevented increases in ROS in the PVN and RVLM. MR- but not AT1aR-siRNA in the SFO prevented the Ang II-induced ROS in the SON. Both MR- and AT1aR-siRNA in the SFO prevented most of the Ang II-induced hypertension as assessed by telemetry. These results indicate that Aldo-MR signaling in the SFO is needed for the activation of Ang II-AT1R-ROS signaling from the SFO to the PVN and RVLM. Activation of Aldo-MR signaling from the SFO to the SON may enhance AT1R dependent activation of pre-sympathetic neurons in the PVN.

Reirradiation and hyperthermia for irresectable locoregional recurrent breast cancer in previously irradiated area: Size matters.

BACKGROUND/PURPOSE: Treatment options for irresectable locoregional recurrent breast cancer in previously irradiated area are limited. Hyperthermia, elevating tumor temperature to 40-45°C, sensitizes radio-and-chemotherapy. Four hundred and fourteen patients treated with reirradiation+hyperthermia (reRT+HT) in the AMC(n=301) and the BVI(n=113), from 1982 to 2005 were retrospectively analyzed for treatment response, locoregional control (LC) and prognostic factors for LC and toxicity. PATIENTS/METHODS: All patients received previous irradiation (median 50 Gy). reRT consisted of 8 × 4 Gy-2/week (AMC) or 12 × 3 Gy-4/week (BVI). Hyperthermia was added once (AMC)/twice (BVI) a week. RESULTS: Overall clinical response rate was 86%. The 3-year LC rate was 25%. The number of recurrence episodes, distant metastases (DM), tumor site, tumor size, time to recurrence and treatment year were significant for LC. Acute ⩾ grade 3 toxicity occurred in 24% of patients. Actuarial late ⩾ grade 3 toxicity was 23% at 3-years. In multivariable analysis reRT fraction dose was significantly related to late ⩾ grade 3 toxicity. CONCLUSION: reRT+HT is an effective curative and palliative treatment option for patients with irresectable locoregional recurrent breast cancer in previously irradiated area. Early referral, treatment of chest wall recurrences ⩽ 5 cm in the absence of distant metastases, provided the highest local control rates. The cumulative effects of past and present treatments should be accounted for by adjusting treatment protocol to minimize toxicity.

Negative feedback-defective PRPS1 mutants drive thiopurine resistance in relapsed childhood ALL.

Relapse is the leading cause of mortality in children with acute lymphoblastic leukemia (ALL). Among chemotherapeutics, thiopurines are key drugs in ALL combination therapy. Using whole-exome sequencing, we identified relapse-specific mutations in the phosphoribosyl pyrophosphate synthetase 1 gene (PRPS1), which encodes a rate-limiting purine biosynthesis enzyme, in 24/358 (6.7%) relapsed childhood B cell ALL (B-ALL) cases. All individuals who harbored PRPS1 mutations relapsed early during treatment, and mutated ALL clones expanded exponentially before clinical relapse. Our functional analyses of PRPS1 mutants uncovered a new chemotherapy-resistance mechanism involving reduced feedback inhibition of de novo purine biosynthesis and competitive inhibition of thiopurine activation. Notably, the de novo purine synthesis inhibitor lometrexol effectively abrogated PRPS1 mutant-driven drug resistance. These results highlight the importance of constitutive activation of the de novo purine synthesis pathway in thiopurine resistance, and they offer therapeutic strategies for the treatment of relapsed and thiopurine-resistant ALL.