1. The tumor microenvironment: problems and opportunities for therapy Nic Denko PhD MD Radiobiology 2013

2. The tumor microenvironment Unique architectural, physiologic and cellular environment Poor perfusion leads to decreased oxygen (hypoxia), decreased nutrients (hypoglycemia), and increased waste products (acidosis All these stresses have compensatory adaptive cellular responses

3. OER can be up to 3 Tannock and Hill BSO 1998 Oxygen is a dose modifying factor

5. Adaptive Biologic Responses Hypoxia: HIF-1 (HIF1a and HIF2a), UPR, SREBP-1, ATF-3, NF-KB Hypoglycemia: HIF-1, Mondo, UPR Acidosis: Sp1 (renal tubule cells), mRNA stability Increased IFP: ???

6. HIF-dependent Gene Induction HRE Transactivation Hypoxia ? PI3K/PTEN/akt ? Other targets? HIFb constitutive Hif1a Inducible Prolyl hydroxylase Elongins VHL P564-OH P402-OH Proteasome Ubi

7. VHL connects hypoxic gene induction to human tumor formation VHL is a classic “two hit” tumor suppressor Loss of VHL leads to constitutive HIF-1 activity Model tumors suggest that this regulation of HIF is a major activity for tumor suppression

8. VHL-resistant HIF is tumorigenic in Renal Cell Cancers Kaelin CC 2002

9. Unfolded proteins generate a complex, three prong response Kaufman JCB 2012

10. Disulfide bond formation requires molecular oxygen Feldman and Koong Mol Can Res 2005 Protein Glycosylation requires glucose

11. How to overcome hypoxia? More oxygen delivery – RBC Mass (transfusion, Epo) – Inhaled oxygen (100% F 1 0 2 ) – Vasorelaxants (hydralazine) Oxygen mimetics (Misonitozole, etanidozole, nimorazole) Hypoxic cytotoxins (MMC, TPZ, AQ4N, Pr104) Less oxygen utilization ?

12. Misonidozole sensitizes hypoxic cells to XRT

13. Miso sensitizes model tumors to single dose XRT treatment 1000 mg/kg Miso pre XRT

14. Development of oxygen mimetics Nimorazole shown to be beneficial in patients with HNC treated with XRT

15. Hypoxic Cytotoxins Tirapazamine Drug is only Toxic in hypoxia

16. TPZ + XRT in vivo TPZ has Low activity as a single agent

17. Oxygen tension is a function of Supply and demand Systemic Circulation Tumor Circulation Supply Demand Can we modify hypoxia from the demand side? Tumor Vasculature

18. 0h 2h 4h 6h 24h d Increased oxygen consumption Causes Increased hypoxia post DCA RKO RKOShHIF Hours post DCA

19. Metabolic (PDHK) inhibitors can enhance TPZ activity in vivo DCA has to be given before TPZ

20. Can we decrease oxygen consumption and decrease hypoxia? Use drugs that decrease mitochondrial function and reduce overall tumor oxygen consumption Measure changes in oxygenation Measure radiosensitization

21. Oxylite A549 tumor MTF response

22. Tumor Microenvironment No microenvironment modifying agents have yet been successful in improving XRT (nimorazole in Europe). HIF1 may be a marginal target, XBP1 may have some efficacy, Hgb increases always make the patient feel better.

23. Which of the following agents has NOT been found to be useful for measuring human tumor hypoxia? A. [18F]- 2-fluoro-2-deoxy-D-glucose (FDG), B. pimonidazole C. [18F]Azomycin Arabinoside (FAZA) D. [64Cu]- Copper dithiosemicarbazone (ATSM) E. [18F]Fluoromisonidazole (FMISO)

24. Pre-treatment correction of anemia with either blood transfusion or epo may be considered in cancer patients. This is based, in part, on which one of the following observations? A. In a German/Swiss, multicenter, randomized, clinical trial, advanced head and neck cancer patients receiving epoetin alfa achieved higher hemoglobin concentrations and longer progression-free survival than placebo. B. Anemia correction has been shown to improve quality of life by reducing fatigue. C. In the Breast Cancer Erythropoietin Survival Trial (BEST), patients with metastatic breast cancer demonstrated improved overall survival when treated with epoetin alpha compared to placebo. D. The activation of hypoxic signaling pathways, including HIF-1α, is associated with resistance to therapy and depends critically on hemoglobin levels

25. Which statement describing tumor vasculature is correct? A. Tumor blood vessels are hyper-permeable, tortuous, and feature haphazard patterns of interconnection, resulting in spatial and temporal heterogeneity of tumor blood flow. B. Tumor blood vessels are hypopermeable, irregular, and densely invested with pericytes resulting in poor diffusion of chemotherapeutics into tumor parenchyma. C. Tumor blood vessels are indistinguishable from normal blood vessels ultrastructurally, however compression caused by proliferating tumor cells leads to vessel collapse and compromised blood flow. D. Tumor blood vessels are dilated, tortuous and have uniformly thick basement membranes, resulting in limited accessibility of chemotherapy agents. E. Tumor blood vessel organization resembles that of normal vessels, however tumor vessels differ ultrastructurally, resulting in poor function and heterogeneous blood flow.

26. The regulation of hypoxia-inducible factor-1α (HIF-1α) by oxygen concentration is best described by which of the following statements? A. Under hypoxic conditions, HIF-1α transcription and translation are upregulated, causing the protein to translocate from the cytosol to the nucleus. B. Under aerobic conditions, the HIF-1α heme moiety becomes oxygenated. This drives a conformational change in the protein limiting DNA binding preventing up-regulation of target genes. C. Under hypoxic conditions, HIF-1α is activated by bioreduction, thereby promoting the up-regulation of target genes. D. Under hypoxic conditions, the HIF-1α heme moiety becomes deoxygenated. This causes a conformational change in the protein, enhancing DNA binding promoting up-regulation of target genes. E. Under aerobic conditions, HIF-1α is hydroxylated by HIF prolyl hydroxylases. This targets the protein for ubiquitination and subsequent proteosomal degradation, thereby preventing the up- regulation of target genes.

27. Methods currently under investigation to monitor the effectiveness of antiangiogenic therapy include all of the following, EXCEPT: A. dynamic MRI/CT and PET imaging B. serial tumor biopsies C. plasma levels of bevacizumab D. VEGF concentration in the urine E. presence of vascular endothelial cells in the peripheral blood