Hypoxic Niche Research Articles

P19 - Mitochondrial Heterogeneity Unveils Properties of Quiescent Hematopoietic Stem Cells

Hematopoietic stem cells (HSCs) produce all blood cells throughout life. This capacity is sustained by HSC quiescence that is compromised with age. Quiescent HSCs are maintained in hypoxic niches, are exquisitely sensitive to levels of reactive oxygen species (ROS), and are thought to rely on glycolysis for their fuel. It remains unknown whether HSC quiescence depends also on mitochondria. Our work suggests that phenotypically defined HSC are highly heterogeneous in their mitochondrial content and function. We find that mitochondrial activity largely segregates quiescent (G0) from cycling-primed (G1) HSCs. Taking advantage of mitochondrial heterogeneity, our work identifies properties specifically associated with quiescent versus primed HSCs. Notably, we find glycolysis to be key in sustaining primed but not quiescent HSCs. Our work also unveils a new mechanism implicated in the maintenance of HSC quiescence that is compromised with age. These findings suggest that mitochondrial heterogeneity further subdivide HSC subpopulations with distinct ROS levels. These findings and how they may relate to our knowledge of ROS regulation of HSC will be discussed.

Mitochondrial Dysfunction and Senescence of Human Cardiac Progenitor Cells Are Prevented by Hypoxic Culture

Rationale: Therapeutic potential of c-Kit+ cardiac progenitor cells (CPCs) is modest in autologous cell therapy clinical trials and could benefit from optimization to maximize functional activity. Early proliferation arrest limits expansion potential of human CPCs in vitro. In contrast, self-renewal is preserved and mitochondrial reactive oxidative species (mtROS) minimized within the CPC hypoxic niche in vivo. Expansion under oxygen tension comparable to the hypoxic niche may therefore improve CPC reparative function.

Emerging Concepts and Therapies for Mucoobstructive Lung Disease.

A spectrum of intrapulmonary airway diseases, for example, cigarette smoke-induced bronchitis, cystic fibrosis, primary ciliary dyskinesia, and non-cystic fibrosis bronchiectasis, can be categorized as "mucoobstructive" airway diseases. A common theme for these diseases appears to be the failure to properly regulate mucus concentration, producing mucus hyperconcentration that slows mucus transport and, importantly, generates plaque/plug adhesion to airway surfaces. These mucus plaques/plugs generate long diffusion distances for oxygen, producing hypoxic niches within adherent airway mucus and subjacent epithelia. Data suggest that concentrated mucus plaques/plugs are proinflammatory, in part mediated by release of IL-1α from hypoxic cells. The infectious component of mucoobstructive diseases may be initiated by anaerobic bacteria that proliferate within the nutrient-rich hypoxic mucus environment. Anaerobes ultimately may condition mucus to provide the environment for a succession to classic airway pathogens, including Staphylococcus aureus, Haemophilus influenzae, and ultimately Pseudomonas aeruginosa. Novel therapies to treat mucoobstructive diseases focus on restoring mucus concentration. Strategies to rehydrate mucus range from the inhalation of osmotically active solutes, designed to draw water into airway surfaces, to strategies designed to manipulate the relative rates of sodium absorption versus chloride secretion to endogenously restore epithelial hydration. Similarly, strategies designed to reduce the mucin burden in the airways, either by reducing mucin production/secretion or by clearing accumulated mucus (e.g., reducing agents), are under development. Thus, the new insights into a unifying process, that is, mucus hyperconcentration, that drives a significant component of the pathogenesis of mucoobstructive diseases promise multiple new therapeutic strategies to aid patients with this syndrome.

Abstract B06: Overcoming hypoxia-induced resistance is essential for effective survivin targeting in acute myeloid leukemia

Abstract Background: Although the clinical outcome of pediatric acute myeloid leukemia (AML) has improved over the past few decades, approximately 30% of patients relapse. The overall survival (OS) rate of pediatric patients with primary refractory or relapsed AML is about 30%. Resistance to therapy presents a great challenge for AML patients. Therefore, there is a critical need to understand mechanisms of resistance, as well as to identify novel therapies that will improve OS. The leukemia-initiating cells reside in special hypoxic niches in the bone marrow. Thus, studying drug response of AML cells within a hypoxic environment is essential for more accurate prediction of patients’ response to therapy. We performed functional drug screening using AML cell lines, which demonstrated a differential response of AML cell lines to the survivin-targeting agent YM155 in normoxia versus hypoxia. Survivin (BIRC5), an inhibitor of apoptosis, is overexpressed in a variety of pediatric blood cancers compared to normal cells. We hypothesize that survivin may be an attractive target for therapy in pediatric AML. The overall objective of this study was to study mechanisms of AML response to YM155 in normoxia and hypoxia and survivin’s potential as a therapeutic target in pediatric AML. Purpose: The aims of the present work were (1) to study BIRC5 expression in publicly available databases of pediatric AML including the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative, and (2) to determine the potential mechanisms of hypoxia-altered response to YM155 in AML cells. Methods and Results: In the present study, the TARGET AML gene-expression profiling data available on Affymetrix human gene 1.1 ST arrays (HuGene 1.1) were used to study BIRC5 expression in comparison to HuGene 1.1 datasets from Gene Expression Omnibus (GEO) containing profiling data from adult AML and normal tissues. BIRC5 expression was significantly higher in pediatric AML in comparison to cytogenetically normal (CN) adult AML, and in relapsed pediatric AML in comparison to matched samples at diagnosis. In order to identify novel targeted therapies in AML we used a panel of AML cell lines to screen a library of 50 compounds in normoxia and hypoxia. The survivin-targeting agent YM155 was found to be one of the most cytotoxic compounds in the majority of AML cell lines in normoxia. However, hypoxia conferred resistance to this drug. The IC50 of YM155 in AML cell lines in normoxia ranged from 1.5 nM-8 µM, with an increase of 2-100 fold in hypoxia (IC50 from 22 nM-14 µM). The Mv4-11 cell line that harbors a FLT3-ITD mutation was selected for further mechanistic studies. Hypoxia conferred resistance to YM155 in Mv4-11 cells through inhibition of apoptosis. In order to identify the underlying molecular mechanisms, we performed RNA-seq of YM155-treated Mv4-11 cells in normoxia and hypoxia, and identified a set of over-represented genes that were validated by quantitative RT-PCR. The mRNA expression of HIF2alpha, p21WAF1, and STAT3 was overexpressed after 9h of YM155 treatment, while PIM2 was downregulated in normoxia but not in hypoxia, in comparison to vehicle-treated control. Preliminary data suggest a role of stem cell genes in hypoxia-induced resistance to YM155. Ongoing studies are directed towards determining the functions of the identified genes in resistance. Conclusion: Our data demonstrate that survivin (BIRC5) is overexpressed in pediatric AML in comparison to adult CN-AML, and may be an attractive target for pediatric AML therapy. Additionally, YM155 is highly cytotoxic to AML cell lines in normoxia; however, for its effective use in the clinic, cotargeting hypoxia-induced resistance factors is essential. Citation Format: David W. Lee, Justin Montoya, Kieran Finch, Jack Wilkinson-Dix, Robert J. Arceci, Daniel H. Wai, Eiman Aleem. Overcoming hypoxia-induced resistance is essential for effective survivin targeting in acute myeloid leukemia [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B06.

Intrabone transplant provides full stemness of cord blood stem cells with fast hematopoietic recovery and low GVHD rate: results from a prospective study

Umbilical Cord Blood (UCB) represents a valid option for patients with hematopoietic malignancies lacking an HLA matched donor. To overcome the limitation of the low stem cell dose of UCB, the intrabone (IB) route has been proposed. We report the results of a prospective study on a poor-prognosis cohort of 23 patients receiving intrabone single UCB transplant (Clinicaltrials.gov NCT00886522). Cumulative incidence of hematological recovery at day 90 was 82 ± 9% (ANC > 0.5 × 109/L) and 70 ± 10% (platelet > 50 × 109/L) and correlated with CD34 + cells in the graft. NRM was 20 ± 9%. No severe aGVHD and only one extensive cGVHD occurred, with fast immune reconstitution. To test the hypothesis that the direct IB injection could affect the expression of stem cells regulatory pathways, CD34 + cells from BM aspirates at day + 10, + 20, + 30, processed in hypoxic conditions mimicking the BM-microenvironment (7%pO2), were studied for the expression of c-Mpl, Notch1 and CXCR4. We found that the expression of c-Mpl in CD34 + cells at day + 10 significantly correlated with hematological recovery. In conclusion, IB-UCB transplant success is associated with low incidence of GVHD and high-speed platelet recovery; intrabone route may preserve full hematopoietic stemness by direct delivery of UCB stem cells into the hypoxic HSC niche.

Therapeutic potency of pharmacological adenosine receptors agonist/antagonist on cancer cell apoptosis in tumor microenvironment, current status, and perspectives.

The hypoxic niche of tumor leads to a tremendous increase in the extracellular adenosine concentration through alteration of adenosine metabolism in the tumor microenvironment (TME). This consequently affects cancer progression, local immune responses, and apoptosis of tumor cells. Regulatory effect of adenosine on apoptosis in TME depends on the cancer cell type, pharmacological characteristics of adenosine receptor subtypes, and the adenosine concentration in the tumor niche. Exploiting specific pharmacological adenosine receptor agonist and antagonist inducing apoptosis in cancer cells can be considered as a proper procedure to control cancer progression. This review summarizes the regulatory role of adenosine in cancer cell apoptosis for a better understanding, and hencebetter management of the disease.

The involvement of the low-oxygen-activated locus of Burkholderia cenocepacia in adaptation during cystic fibrosis infection

Chronic infection with opportunistic pathogens including Burkholderia cepacia complex (Bcc) is a hallmark of cystic fibrosis (CF). We investigated the adaptive mechanisms facilitating chronic lung infection in sequential Bcc isolates from two siblings with CF (P1 and P2), one of whom also experienced intermittent blood-stream infections (P2). We previously showed increased lung cell attachment with colonisation time in both P1 and P2. WGS analysis confirmed that the isolates are closely related. Twelve genes showed three or more mutations, suggesting these were genes under selection. Single nucleotide polymorphisms (SNVs) in 45 regulatory genes were also observed. Proteomic analysis showed that the abundance of 149 proteins increased over 61-months in sputum isolates, and both time- and source-related alterations in protein abundance between the second patient’s isolates. A consistent time-dependent increase in abundance of 19 proteins encoded by a low-oxygen-activated (lxa) locus was observed in both sets of isolates. Attachment was dramatically reduced in a B. cenocepacia K56-2Δlxa-locus deletion mutant, further indicating that it encodes protein(s) involved in host-cell attachment. Time-related changes in virulence in Galleria mellonella or motility were not observed. We conclude that the lxa-locus, associated with anoxic persistence in vitro, plays a role in host-cell attachment and adaptation to chronic colonization in the hypoxic niche of the CF lung.

Uncoupling Warburg effect and stemness in CD133+ve cancer stem cells from Saos-2 (osteosarcoma) cell line under hypoxia.

Cancer stem cells (CSCs) which are known to be residing deep inside the core of the tumor in its hypoxia niche is responsible for relapse of cancers. Owing to this hypoxic niche, the residing CSCs simultaneously fuel their stemness, cancerous and drug resistance properties. Attributes of CSCs are still not properly understood in its hypoxia niche. Addressing this, we sorted CSCs from Saos-2 (osteosarcoma) cell line using CD133 antibody. The CD133+ve CSCs exhibited quiescent cell proliferation in DNA doubling, Ca2+ signaling and cell cycle analysis. CD133+ve CSCs exhibited increased production of ATP and lactate dehydrogenase (LDH) activity under hypoxia. CD133+ve cells exhibited decreased glucose uptake compared to ATP levels under hypoxia. Moreover, there was only negligible LDH activity in CD133+ve cells under normoxia which do not rely on Warburg effect. Stemness markers (such as c-Myc, SOX2, Oct4 and TERT), metastasis marker (CD44) and drug resistance marker (ABCG2) were highly expressed in CD133+ve cells. In summary, both CD133+ve/-ve cells of Saos-2 (osteosarcoma) cell line did not exhibit Warburg effect under normoxic condition. Moreover, this significantly indicates an uncoupling between stemness and Warburg effect in CD133+ve. This work provides a novel insight into the metabolic and functional features of CSCs in a hypoxic environment which could open new avenues for therapeutic strategies aimed to target CSCs.

RNA binding protein HuR regulates extracellular matrix gene expression and pH homeostasis independent of controlling HIF-1α signaling in nucleus pulposus cells

Nucleus pulposus (NP) cells reside in the hypoxic niche of the intervertebral disc. Studies have demonstrated that RNA-binding protein HuR modulates hypoxic signaling in several cancers, however, its function in the disc is unknown. HuR did not show cytoplasmic translocation in hypoxia and its silencing did not alter levels of Hif-1α or HIF-targets in NP cells. RNA-Sequencing data revealed that important extracellular matrix-related genes including several collagens, MMPs, aggrecan, Tgf-β3 and Sdc4 were regulated by HuR. Further analysis of HuR-silenced NP cells confirmed that HuR maintained expression of these matrix genes. We confirmed decreased levels of secreted collagen I and Sdc4 and increased pro-MMP13 in HuR-knockdown cells. In addition, messenger ribonucleoprotein immunoprecipitation demonstrated HuR binding to Tgf-β3 and Sdc4 mRNAs. Interestingly, while HuR bound to Hif-1α and Vegf mRNAs, it was clear that compensatory mechanisms sustained their expression when HuR was silenced. Noteworthy, despite the presence of multiple HuR-binding sites and reported interaction in other cell types, HuR showed no binding to Pgk1, Eno1, Pdk1 and Pfkfb3 in NP cells. Metabolic studies showed a significant decrease in the extracellular acidification rate (ECAR) and mitochondrial oxygen consumption rate (OCR) and acidic pH in HuR-silenced NP cells, without appreciable change in total OCR. These changes were likely due to decreased Ca12 expression in HuR silenced cells. Taken together, our study demonstrates for the first time that HuR regulates extracellular matrix (ECM) and pH homeostasis of NP cells and has important implications in the maintenance of intervertebral disc health.

Abstract 468: Mitochondrial Dysfunction and Senescence of Human Cardiac Progenitor Cells Are Prevented by Hypoxic Culture

Rationale: Therapeutic potential of c-Kit + cardiac progenitor cells (CPCs) is modest in autologous cell therapy clinical trials and could benefit from optimization to maximize functional activity. Early proliferation arrest limits expansion potential of human CPCs in vitro . In contrast, self-renewal is preserved and mitochondrial reactive oxidative species (mtROS) minimized within the CPC hypoxic niche in vivo . Expansion under oxygen tension comparable to the hypoxic niche may therefore improve CPC reparative function. Hypothesis: Senescence of CPCs derived from heart failure patients is suppressed by dampening mtROS through long-term hypoxic culture. Methods and Results: Environmental oxygen was maintained with constant hypoxia (1% O 2 ; H-CPCs) or atmospheric oxygen tension (20 - 21% O 2 ; N-CPCs) for isolation and expansion of CPCs from patients undergoing left ventricular assist device implantation. A 4-fold increase in clonogenesis and 73% fewer senescence-associated β-Gal-positive cells were found in H-CPCs. Oxidative stress and DNA damage were also reduced with 80% lower malondialdehyde content and significantly fewer nuclear γ-H2AX foci in H-CPCs. Accumulation of dysfunctional mitochondria was revealed in N-CPCs through functional metabolic analyses. Basal (-59%), maximal (-50%), and ATP production-coupled (-62%) oxygen consumption rates were significantly lower in N-CPCs, despite upregulation of electron transport chain subunits and 72% greater mitochondrial DNA content than H-CPCs. MtROS was elevated, and large mitochondria with swollen morphology were found by TEM in N-CPCs, confirming mitochondrial damage. Key indicators of mitochondrial function including NAD + /NADH ratio and autophagic flux were preserved in H-CPCs. Conclusions: Development of mitochondrial dysfunction and senescence are consequences of normoxic CPC expansion. CPC senescence is delayed by preserving mitochondrial health and limiting oxidative damage through hypoxic culture.

High-Throughput Flow Cytometry Identifies Small-Molecule Inhibitors for Drug Repurposing in T-ALL

Kinase inhibitors have dramatically increased patient survival in a multitude of cancers, including hematological malignancies. However, kinase inhibitors have not yet been integrated into current clinical trials for patients with T-cell-lineage acute lymphoblastic leukemia (T-ALL). In this study, we used a high-throughput flow cytometry (HTFC) approach to test a collection of small-molecule inhibitors, including 26 FDA-approved tyrosine kinase inhibitors in a panel of T-ALL cell lines and patient-derived xenografts. Because hypoxia is known to cause resistance to chemotherapy, we developed a synthetic niche that mimics the low oxygen levels found in leukemic bone marrow to evaluate the effects of hypoxia on the tested inhibitors. Drug sensitivity screening was performed using the Agilent BioCel automated liquid handling system integrated with the HyperCyt HT flow cytometry platform, and the uptake of propidium iodide was used as an indication of cell viability. The HTFC dose–response testing identified several compounds that were efficacious in both normal and hypoxic conditions. This study shows that some clinically approved kinase inhibitors target T-ALL in the hypoxic niche of the bone marrow.

Tissue-Penetrating, Hypoxia-Responsive Echogenic Polymersomes For Drug Delivery To Solid Tumors.

Hypoxia in solid tumors facilitates the progression of the disease, develops resistance to chemo and radiotherapy, and contributes to relapse. Due to the lack of tumor penetration, most of the reported drug carriers are unable to reach the hypoxic niches of the solid tumors. We have developed tissue-penetrating, hypoxia-responsive echogenic polymersomes to deliver anticancer drugs to solid tumors. The polymersomes are composed of a hypoxia-responsive azobenzene conjugated and a tissue penetrating peptide functionalized polylactic acid-polyethylene glycol polymer. The drug-encapsulated, hypoxia-responsive polymersomes substantially decreased the viability of pancreatic cancer cells in spheroidal cultures. Under normoxic conditions, polymersomes were echogenic at diagnostic ultrasound frequencies but lose the echogenicity under hypoxia. In-vivo imaging studies with xenograft mouse model further confirmed the ability of the polymersomes to target, penetrate, and deliver the encapsulated contents in hypoxic pancreatic tumor tissues.

Cysteine cathepsins B, X and K expression in peri-arteriolar glioblastoma stem cell niches

Glioblastoma (GBM) is the most lethal brain tumor also due to malignant and therapy-resistant GBM stem cells (GSCs) that are localized in protecting hypoxic GSC niches. Some members of the cysteine cathepsin family of proteases have been found to be upregulated in GBM. Cathepsin K gene expression is highly elevated in GBM tissue versus normal brain and it has been suggested to regulate GSC migration out of the niches. Here, we investigated the cellular distribution of cathepsins B, X and K in GBM tissue and whether these cathepsins are co-localized in GSC niches. Therefore, we determined expression of these cathepsins in serial paraffin sections of 14 human GBM samples and serial cryostat sections of two samples using immunohistochemistry and metabolic mapping of cathepsin activity using selective fluorogenic substrates. We detected cathepsins B, X and K in peri-arteriolar GSC niches in 9 out of 16 GBM samples, which were defined by co-expression of the GSC marker CD133, the niche marker stromal-derived factor-1α (SDF-1α) and smooth muscle actin as a marker for arterioles. The expression of cathepsin B and X was detected in stromal cells and cancer cells throughout the GBM sections, whereas cathepsin K expression was more restricted to arteriole-rich regions in the GBM sections. Metabolic mapping showed that cathepsin B, but not cathepsin K is active in GSC niches. On the basis of these findings, it is concluded that cathepsins B, X and K have distinct functions in GBM and that cathepsin K is the most likely GSC niche-related cathepsin of the three cathepsins investigated.

Metabolic and Physiologic Imaging Biomarkers of the Tumor Microenvironment Predict Treatment Outcome with Radiation or a Hypoxia-Activated Prodrug in Mice.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by hypoxic niches that lead to treatment resistance. Therefore, studies of tumor oxygenation and metabolic profiling should contribute to improved treatment strategies. Here, we define two imaging biomarkers that predict differences in tumor response to therapy: (i) partial oxygen pressure (pO2), measured by EPR imaging; and (ii) [1-13C] pyruvate metabolism rate, measured by hyperpolarized 13C MRI. Three human PDAC xenografts with varying treatment sensitivity (Hs766t, MiaPaCa2, and Su.86.86) were grown in mice. The median pO2 of the mature Hs766t, MiaPaCa2, and Su.86.86 tumors was 9.1 ± 1.7, 11.1 ± 2.2, and 17.6 ± 2.6 mm Hg, and the rate of pyruvate-to-lactate conversion was 2.72 ± 0.48, 2.28 ± 0.26, and 1.98 ± 0.51 per minute, respectively (n = 6, each). These results are in agreement with steady-state data of matabolites quantified by mass spectroscopy and histologic analysis, indicating glycolytic and hypoxia profile in Hs766t, MiaPaca2, and Su.86.86 tumors. Fractionated radiotherapy (5 Gy × 5) resulted in a tumor growth delay of 16.7 ± 1.6 and 18.0 ± 2.7 days in MiaPaca2 and Su.86.86 tumors, respectively, compared with 6.3 ± 2.7 days in hypoxic Hs766t tumors. Treatment with gemcitabine, a first-line chemotherapeutic agent, or the hypoxia-activated prodrug TH-302 was more effective against Hs766t tumors (20.0 ± 3.5 and 25.0 ± 7.7 days increase in survival time, respectively) than MiaPaCa2 (2.7 ± 0.4 and 6.7 ± 0.7 days) and Su.86.86 (4.7 ± 0.6 and 0.7 ± 0.6 days) tumors. Collectively, these results demonstrate the ability of molecular imaging biomarkers to predict the response of PDAC to treatment with radiotherapy and TH-302.Significance: pO2 imaging data and clinically available metabolic imaging data provide useful insight into predicting the treatment efficacy of chemotherapy, radiation, and a hypoxia-activated prodrug as monotherapies and combination therapies in PDAC tumor xenograft models. Cancer Res; 78(14); 3783-92. ©2018 AACR.

Abstract 5476: Inhibiting lactate transporters MCT-1 and MCT-4 target hypoxic HNSCC cells and sensitize them to metformin

Abstract Head and neck squamous cell carcinoma (HNSCC) is treated primarily through a combination of surgery and radiation therapy. The problem of radioresistance, however, persists and requires new approaches to overcome it. Tumor hypoxia has been shown to be a driver of radioresistance, thereby prompting the targeting of the hypoxic niche, known to be highly glycolytic. Two key components of the hypoxic metabolic profile are lactate transporters MCT-1 and MCT-4, which sustain hypoxia-driven lactate production in cells. We hypothesize that inhibition of MCT-1 and MCT-4 will selectively target the growth of hypoxic tumor cells and potentially exert a synergistic effect with the antidiabetic drug metformin, a known inhibitor of mitochondrial respiration that has been shown to improve the radiation response. To study this, we employed CRISPR-Cas9 to genetically ablate both MCT-1 and MCT-4 in an HNSCC cell line. Validation of double knockout (DKO) cells was done through immunoblotting and sequencing. Cells were grown under conditions of 21% O2 (normoxia) in a regular CO2 incubator or 0.2% O2 (hypoxia) in an H45 HypOxystation® hypoxia chamber. Cell proliferation was measured under normoxia and hypoxia through the use of IncuCyte automated imaging while the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of cells were measured using a Seahorse™ XF analyzer. Metabolic profiling of the DKO cells showed a higher rate of oxygen consumption and a decreased ability of exporting lactate, indicating a metabolic shift when lactate transport is impaired. The loss of MCT-1 and MCT-4 by themselves did not significantly alter cell proliferation under normoxic conditions, but disruption of both transporters concurrently significantly altered growth under normoxia. When exposed to hypoxia, proliferation of the DKO cells was completely halted. In addition, MCT-1/MCT-4 double knockouts showed greater inhibition of cell growth under metformin treatment than either single knockouts or wild-type cells. Significantly lower doses of metformin, which had no effect on the proliferation of wild-type or single knockout cells, were capable of impairing the growth of DKO cells under hypoxia. In conclusion, our study shows that inhibition of lactate export has a profound effect on cell growth under hypoxic conditions in vitro. Moreover, loss of lactate export enhances the sensitivity of HNSCC cells to metformin and could constitute a new way of targeting the hypoxic niche with the purpose of leading to better treatment outcomes. Citation Format: Pedro H. Boasquevisque, Verena Schoeneberger, Laura Caporiccio, Ravi Vellanki, Marianne Koritzinsky, Bradly G. Wouters. Inhibiting lactate transporters MCT-1 and MCT-4 target hypoxic HNSCC cells and sensitize them to metformin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5476.

Abstract 2427: Targeting intracellular pH regulation as a novel approach to overcome hypoxia-mediated drug resistance in FLT3/ITD mutated AML

Abstract Background: FLT3/ITD+ AML is a highly aggressive form of leukemia that carries a dismal prognosis. Evidence suggests that this poor outcome is due to the survival of a subset of leukemic cells (LCs) that elude therapy and survive in hypoxic niches of the bone marrow (BM). Effective targeting of LCs under hypoxic conditions is thus critical to cure AML. Our study examines the anti-leukemic potential of targeting Carbonic anhydrase-IX (CA9), a transmembrane protein which functions to maintain a neutral intracellular pH (pHi) under hypoxic stress conditions, against AML cells. Targeting CA9 is of particular interest as its expression is confined to only a few normal tissues and may therefore show relatively few side effects compared to standard therapies. Methods: Molm14 (M14) and primary cells (PC) from FLT3/ITD+ AML patients were incubated under normoxic (21% O2) and hypoxic (1% O2) conditions in the presence or absence of Quizartinib (Q), Cytarabine (Cy) or the CA9 inhibitor FC531 (FC). After 48h proliferation and apoptosis were determined per MTT assays, annexin V/PI staining and FACS analysis. Cells were assessed for CA9 mRNA expression as well as pHi levels via RT-PCR and fluorescent imaging, respectively. FLT3/ITD+ AML xenografts were generated by injecting M14 cells in the tail vein of NSG mice. Tissue samples were fixed in 10% NBF, embedded in paraffin and stained with H&E and CA9 for pathologic evaluation. Results: Cy and Q were significantly less effective against M14 cells under 1% compared to 21% O2. CA9 mRNA was upregulated in M14 cells (4.5±0.9-fold; n=4, p<.01) and in FLT3/ITD+ AML PCs from newly diagnosed (n=5) and relapsed/ refractory (n=3) patients (up to 529- and 73-fold, respectively) under 1% compared to 21% O2. Immuno-histochemical staining of the BM and spleen obtained from AML xenografts showed multifocal CA9 staining of LCs that was localized on the cell membrane. Under hypoxic conditions, FC but not Cy or Q, significantly induced apoptosis and showed dose-dependent growth inhibitory effects against M14 cells (n=3). In addition, treatment of M14 cells with FC under 1% O2 resulted in strong pHi acidification (ΔpH=-2.7±0.6, p<.05, n=3). Moreover, FC combined with Cy synergistically inhibited M14 cell growth in vitro and effectively eliminated LCs from the BM and spleen compared to Cy only treated mice. Accordingly, spleen size and weight were significantly reduced in mice treated with FC and Cy compared to single agent Cy (n=3). Conclusions: 1. Hypoxia blunts the effects Cy and Q in FLT3/ITD+ AML. 2. CA9 is induced in FLT3/ITD+ AML cells under hypoxic conditions. 3. The CA9 inhibitor FC confers anti-leukemic activity against FLT3/ITD+ AML cells under hypoxic conditions via pHi acidification and acts synergistically with Cy. 4. CA9 inhibition has the potential to target LCs residing in hypoxic niches and may be of value as an adjunct to chemotherapy for FLT3/ITD+AML therapy. Citation Format: Fangli Chen, Adriana L. Rogozea, Xue Wu, George Sandusky, Mircea Ivan, Heiko Konig. Targeting intracellular pH regulation as a novel approach to overcome hypoxia-mediated drug resistance in FLT3/ITD mutated AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2427.

Abstract 4959: CP-506, a next generation hypoxia-activated prodrug, as promising novel anti-cancer therapeutic

Abstract Hypoxia is a characteristic of many solid tumors and is defined as a low level or absence of oxygen due to an insufficient vascularization of the tumor or a transient blockage of blood vessels. Hypoxia activates a survival response within the tumor cells driving cancer progression and is associated with poor prognosis. Hypoxia-activated prodrugs (HAPs) are anti-neoplastic agents that can solely be activated in hypoxic areas allowing the targeted delivery of cytotoxic compounds in the hypoxic tumor niches. Convert Pharmaceuticals is developing CP-506, a novel generation of HAP presenting a strong bystander effect. A key step in the activation of CP-506 is its conversion by one-electron reductases to an oxygen-sensing intermediate. Under normoxic conditions, the intermediate is rapidly re-oxidized to the pro-drug. However, under severe hypoxic conditions, further reduction steps lead to the generation of the active cytotoxic metabolite, CP-506M. In an in vitro cytotoxicity assay, CP-506 significantly inhibited the viability of several tumor cell lines specifically under anoxic conditions. In spheroid models, CP-506 demonstrates a strong bystander effect i.e. the ability of the active metabolite to locally diffuse beyond the hypoxic core and induce tumor cell death. CP-506 was administered intraperitoneally at a dose of 600 mg/kg once a day for 5 consecutive days and showed a significant inhibition of tumor growth in four tumor xenograft models (triple negative breast cancer, lung and pancreatic cancers). In all tumor models, CP-506 treatment significantly increased the survival of treated mice assessed by the time to reach four times the start volume as a surrogate endpoint for survival. Different dosing regimens for CP-506 were investigated and all demonstrated a significant tumor growth inhibition compared to the vehicle group. The most profound anti-tumor effect was observed for the repeated cycles of daily consecutive administrations. Finally, no cumulative toxicity was observed for any of the repeated dosing schedules as indicated by bodyweight change. The successful development of CP-506 will require combination with treatments targeting well-oxygenated cells and the careful selection of patients predicted to benefit from a HAP treatment. To this end, we are currently identifying key parameters (presence of tumor hypoxia, expression of reductases and DNA repair capacity) in the activation of CP-506 to define predictive gene signatures in liquid biopsies enabling patient selection. Our current pre-clinical data strongly suggest that CP-506 is a potent, highly selective HAP with a favorable pharmacokinetic profile and the addition of predictive gene signatures or imaging biomarkers for patient selection holds great promise for the treatment of cancer. Citation Format: sophie thiolloy, Sofie Deschoemaeker, Nicolas Ongenae, Julie Gilissen, Ludwig Dubois, Ala Yaromina, Amir Ashoorzadeh, Jeff Smaill, Adam Patterson, Philippe Lambin, Arne Heyerick. CP-506, a next generation hypoxia-activated prodrug, as promising novel anti-cancer therapeutic [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4959.

WNK1 kinase and its partners Akt, SGK1 and NBC-family Na+/HCO3- cotransporters are potential therapeutic targets for glioblastoma stem-like cells linked to Bisacodyl signaling.

Glioblastoma is a highly heterogeneous brain tumor. The presence of cancer cells with stem-like and tumor initiation/propagation properties contributes to poor prognosis. Glioblastoma cancer stem-like cells (GSC) reside in hypoxic and acidic niches favoring cell quiescence and drug resistance. A high throughput screening recently identified the laxative Bisacodyl as a cytotoxic compound targeting quiescent GSC placed in acidic microenvironments. Bisacodyl activity requires its hydrolysis into DDPM, its pharmacologically active derivative. Bisacodyl was further shown to induce tumor shrinking and increase survival in in vivo glioblastoma models. Here we explored the cellular mechanism underlying Bisacodyl cytotoxic effects using quiescent GSC in an acidic microenvironment and GSC-derived 3D macro-spheres. These spheres mimic many aspects of glioblastoma tumors in vivo, including hypoxic/acidic areas containing quiescent cells. Phosphokinase protein arrays combined with pharmacological and genetic modulation of signaling pathways point to the WNK1 serine/threonine protein kinase as a mediator of Bisacodyl cytotoxic effect in both cell models. WNK1 partners including the Akt and SGK1 protein kinases and NBC-family Na+/HCO3− cotransporters were shown to participate in the compound’s effect on GSC. Overall, our findings uncover novel potential therapeutic targets for combatting glioblastoma which is presently an incurable disease.

PH-Sensitive Multiligand Gold Nanoplatform Targeting Carbonic Anhydrase IX Enhances the Delivery of Doxorubicin to Hypoxic Tumor Spheroids and Overcomes the Hypoxia-Induced Chemoresistance.

Hypoxia is a common feature of solid tumors contributing to resistance to chemotherapy. Selective delivery of chemotherapeutic drugs to hypoxic tumor niche remains an unsolved issue. For this purpose, we constructed a gold nanoplatform targeting carbonic anhydrase IX (CA IX) epitope, which is overexpressed in hypoxic tumor cells versus in normal tissues. We designed compatible low-molecular weight carbonic anhydrase inhibitor (CAI) ligands and doxorubicin (Dox) ligands and optimized protocols for efficient decoration of gold nanoparticles (Au NPs) to achieve both good targeting ligand density and optimum drug loading, while preserving colloidal stability. The optimized Dox-HZN-DTDP@Au NPs-LA-PEG2000-CAI (THZN) nanoplatform was proved to be very efficient toward killing HT-29 tumor cells, especially under hypoxic conditions, as compared with the nontargeting nanoplatform. This also mediated the effective release of doxorubicin in the lysosomes following internalization, as revealed by confocal microscopy. Furthermore, using tumor spheroids as a representative model for hypoxic solid tumors, our THZN nanoplatform enhanced the selective delivery of doxorubicin up to 2.5 times and minimized chemoresistance, showing better tumor drug penetration as compared to that in free drug treatment. Our technology is the first CA IX-targeting gold nanoplatform for efficient delivery of doxorubicin to hypoxic tumors in a controlled fashion, with the perspective to improve the therapy of solid tumors and minimize chemoresistance.

Intratumoral heterogeneity of oxygen metabolism and neovascularization uncovers 2 survival-relevant subgroups of IDH1 wild-type glioblastoma.

The intratumoral heterogeneity of oxygen metabolism in combination with variable patterns of neovascularization (NV) as well as reprogramming of energy metabolism affects the landscape of tumor microenvironments (TMEs) in glioblastoma. Knowledge of the hypoxic and perivascular niches within the TME is essential for understanding treatment failure. Fifty-two patients with untreated glioblastoma (isocitrate dehydrogenase 1 wild type [IDH1wt]) were examined with a physiological MRI protocol including a multiparametric quantitative blood oxygen level dependent (qBOLD) approach and vascular architecture mapping (VAM). Imaging biomarker information about oxygen metabolism (mitochondrial oxygen tension) and neovascularization (microvascular density and type) were fused for classification of 6 different TMEs: necrosis, hypoxia with/without neovascularization, oxidative phosphorylation (OxPhos), and glycolysis with/without neovascularization. Association of the different TME volume fractions with progression-free survival (PFS) was assessed using Kaplan-Meier analysis and Cox proportional hazards models. A common spatial structure of TMEs was detected: central necrosis surrounded by tumor hypoxia (with defective and functional neovasculature) and different TMEs with a predominance of OxPhos and glycolysis for energy production, respectively. The percentage of the different TMEs on the total tumor volume uncovered 2 clearly different subtypes of glioblastoma IDH1wt: a glycolytic dominated phenotype with predominantly functional neovasculature and a necrotic/hypoxic dominated phenotype with approximately 50% of defective neovasculature. Patients with a necrotic/hypoxic dominated phenotype showed significantly shorter PFS (P = 0.035). Our non-invasive mapping approach allows for classification of the TME and detection of tumor-supportive niches in glioblastoma which may be helpful for both clinical patient management and research.