Compensatory Angiogenesis and Tumor Refractoriness Prof. Rajesh N Gacche Tumor Biology Laboratory, School of Life Sciences SRTM Univeristy, Nanded, India (MS)

Angiogenesis is the formation of new blood vessels from preexisting one

Structure of vessels and capillaries Monocellular layer of endothelial cellsSmall artery: Capillary: endothelial cell, basal lamina, pericytes

Angiogenesis is regulated by endogenous activators and Inhibitors

Anti-Angiogenic factors Normal angiogenesis Pro-Angiogenic factors Anti-Angiogenic factors Excessive angiogenesis Pro-Angiogenic factors Anti-Angiogenic factors Normal angiogenesis Pro-Angiogenic factors Anti-Angiogenic factors Insufficient angiogenesis

MMPs (Matrix Metallo- Proteinases) VEGF, FGF, PGF, IGF, Angp, EGF, HGF, HIF, TGF, IL-3, IL-8, Ang VEGF, FGF, PGDF, PGF, IGF, Angp, EGF, HGF, HIF, TGF, TNF, IL-3, IL-8, Ang VEGF, PGF, HIF-1 1. Activation 2. Basement membrane degradation 3. Migration 4. Differentiation / Proliferation / Tube formation 5. Stabilization / Maturation VEGF, PGDF, IGF, Angp Tumor Cytokines, FGF Induce Expression of VEGF IL-1, IL-6 Regulate Process of Tumor Angiogenesis

VEGFR-Family FGFR PDGFR PLCγ IP3 NOSIII NO Vasodilation/ Permeability DAG PKC Cell Proliferation SHC FAK Paxillin Cell Migration PI3K Cell Survival PI3K RhoA Vav2 Vav1 Actin, Stress fibres & Adhesion Rac1 Por1Por2 Lamellipodia / Filopoda Formation CDC42 N-WASP A n g i o g e n e s i s PI3K PIP2 PIP3 PIP2BAD Caspase 9 Tie2/ Tek PLCγ PIP3 RAS cRAF MEK 1/2 ERK 1/2 IP3 CamK II PI3K AKT Pathway RAS CDC42RAC1 Regulation of Cytoskeleton c-MET VEGF- Family CDC42 Pathway RAC1 Pathway AKT Pathway FGF Ang-1,2,3 PDGF HGF XL880, TAK-701, Relotumumab Brivanib, Nintedanib, Pentosan- polysulfate Avastin, Aflibercept, Pegaptanib, Pazopanib, Ranibizumab, Sunitinib Sorafenib Regorafenib, Ponatinib

3D structural information of angiogenic proteins has profoundly influenced the philosophy of drug design and development. There have been major and striking advances in protein crystallography. Structures solved by protein crystallography are exceptionally valuable and forms foundation for effective ligand design. Structural knowledge can be effectively utilized in developing better therapeutic agents for modulation of angiogenesis in cancer therapy. Targeting Tumor Angiogenesis for designing novel drugs

VEGF-B VEGFR-1 VEGF-C VEGFR-2 These interactions have opened novel therapeutic avenues to study the role of VEGFR-1-specific ligand in angiogenesis- mediated pathologies. These contacts can be utilized in generating peptide mimetic inhibitor molecules that can modulate VEGF-C interaction with VEGFR-2 These contacts can be utilized in generating peptide mimetic inhibitor molecules that can modulate VEGF-C interaction with VEGFR-2 Hydrogen bonds Hydrophobic contacts

Interactions at interface of FGF-1::FGFR opens new avenues for rational drug design targeting FGF1-induced angiogenesis and cell proliferation. Interactions at interface of FGF-1::FGFR opens new avenues for rational drug design targeting FGF1-induced angiogenesis and cell proliferation. FGFR FGF-2 FGFR FGF-1 These interactions provide a structural insight to design therapeutic agents that can target FGF-2::FGFR interactions. These interactions provide a structural insight to design therapeutic agents that can target FGF-2::FGFR interactions.

These interactions hold a key to design strategy for modulation of PGF- VEGFR-1 interaction. Such biochemical communications can be efficiently utilized to modulate PGDF β -receptor interaction in therapeutic context. Such biochemical communications can be efficiently utilized to modulate PGDF β -receptor interaction in therapeutic context. PGDFR PGDF- β VEGFR-1 PGF

These contacts at ATP binding site can be utilized to develop therapeutically relevant agents targeting Tie2K activity. Interactions at IGFBP and IGF can guide development of interaction based inhibitors. IGF IGFBP Tie2K

These interactions at HGF::c-Met thus provide an opportunity to selectively modulate HGF activity as antagonist for cancer therapy. These interactions at HGF::c-Met thus provide an opportunity to selectively modulate HGF activity as antagonist for cancer therapy. Biochemical interactions at EGF::EGFR interface possess enormous potential to develop contact based therapeutic agents. EGF EGFR HGF C-Met

All these interactions at TGF::EGFR possess lot of potential to be therapeutically targeted. All these interactions at TGF::EGFR possess lot of potential to be therapeutically targeted. Interactions at HIF- CBP complex can be extensively used to develop small molecule transcriptional modulators. Interactions at HIF- CBP complex can be extensively used to develop small molecule transcriptional modulators. HIF CBP

More about structural opportunities for developing anti-angiogenic agents

Drugs FDA Approval Improvement in PFS (Months) Improvement in OS (Months) Bevacizumab metastatic colorectal cancer (with chemotherapy) metastatic nonsquamous NSCLC (with chemotherapy) advanced cervical cancer (with chemotherapy) Sunitinibmetastatic RCC (Renal cell carcinoma)64.6 Sorafenibmetastatic RCC2.7NS Pazopanibmetastatic RCC5NA Vandetanibadvanced medullary thyroid cancer6.2NA Axitinibadvanced RCC2NA Regorafenib chemorefractory metastatic colorectal cancer Aflibercept chemorefractory metastatic colorectal cancer Cabozantinibadvanced medullary thyroid cancer7.2NS Ramucirumabmetastatic gastric and GEJ cancers Source: Rakesh Jain, Cancer Cell 26, November 10, 2014

Clinical Research in Angiogenesis Inhibitors as on 1 st Nov Clinical trials are registered 1445 Trials have been completed (41 %) 356 Trials have been Terminated (10 %) 89 Trials have been withdrawn (2.5 %) 14 Trials have been suspended (0.4 %) Source: Clinical Trials.gov

Arguments ? Targeting Tumor Angiogenesis: a Right target or a Wrong Choice ? Why the tumors growth is more aggressive after drug holidays ? Why there is evolving drugs resistance towards anti-angiogenic agents ? Does the compensatory angiogenic mechanisms is the major factors in limiting the efficacy of anti-angiogenic therapy ?

Targeting tumor angiogenesis: an attractive target with emerging challenges Pathophysiologic al point of view Without neovascularisation No tumor growth beyond a size of 2 mm No metastasis pharmacological point of view How will you supply an anti-cancer drugs to the tumor without an appropriate blood supply? Targeting Angiogenesis: Right target or a Wrong Choice ?

‘Normalization of tumor vasculature’: a new paradigm by Prof. Rakesh Jain Blood vessels of tumor are more complex, dilated, tortuous, hyperpermeable and disorganized This makes the access of drug molecules difficult to reach every cell of tumor body. Instead of killing the entire tumor vessels, it is imperative to normalize (organized vessel complex) it initially? Appropriate doses of anti-antiangiogenic drugs has been shown to normalize the vessels.

Goel S et al. Physiol Rev 2011;91:

Inbuilt threats of targeting tumor angiogenesis At present anti-angiogenic agents can not discriminate between physiological and pathological angiogenesis. Hence, hampers normal angiogenesis. Anti-angiogenic agents lack efficacy due to prevalence of compensatory angiogenesis pathways. Off-target toxicities unrelated to blockade of physiological angiogenesis………another big issue!!

Conspiracy of Compensatory Angiogenesis in acquired drug resistance A. VEGF dependent B. VEGF independent: – FGF, PDGF, Angiopetins, Ephrins etc – DLL4-Notch Signalling C. Myeoloid & Stromal/Tumor Cell mediated angiogenic reprogramming. D. Angiogenesis independent remodeling mechanisms like vascular mimicry, vessel cooption and in intussusceptive angiogenesis

Source: Gacche RN, 2015,Oncogenesis (Nature)

VEGF-axis dependent and non-VEGF mediated mechanisms of resistance to anti-angiogenic therapies Source: Gacche RN, 2015,Oncogenesis (Nature)

VEGFR-2 Vasodilatation/ Permeability Cell Proliferation Cell Migration Cell Survival A n g i o g e n e s i s VEGF – A Signaling VEGFR-3 Avastin VEGF-A VEGF-D VEGF-A By pass Proteolytic cleavage VEGFR-3 Sustained Angiogenesis in VEGFR-2 Inhibition State VEGF -C PlGF FGFR FGFR Signaling FGF Synergistic activity Synergistic Inhibition DLL-4 NOTCH NOTCH Signaling Up regulate anti-angiogenic resistance in VEGFA targeted therapies Endothelial cell VEGF bypass pathways

Source: Gacche RN, 2015,Oncogenesis (Nature)

Conclusions Based on the present clinical and epidemiological literature it is clear that the future settings of targeting tumor angiogenesis should customize more on inhibiting the compensatory angiogenic pathways/factors so as to improve the efficacy of anti-angiogenic agents. Developing anti-tumor agents hitting multiple targets are more appreciated in the midst of evolving resistance of cancer cells towards present day anticancer drugs

In silico work of di-, tri-, tetra-, and penta- hydroxy substituted flavones Quantum chemical descriptors Nanded Thank you