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  • PI3K pathway is an exciting and promising therapeutic target for cancer
    It’s known to play important roles in tumor cell proliferation and survival, and it’s been shown to be an important mediator of resistance to targeted therapies such as EGFR inhibitors and conventional cytotoxic agents.
    mTOR/Raptor drives a feedback loop, mediated via IRS-1 phosphorylation that normally keeps PI3K activity in check.
    One consequence of mTOR/raptor inhibition by rapamycin and rapamycin analogs is alleviation of this negative feedback loop resulting in activation of PI3K and subsequent activation of AKT.
    Therefore, simultaneously targeting both PI3K and mTOR has the potential to simultaneously inhibit both upstream and downstream signaling in the pathway.
  • PI3K pathway is an exciting and promising therapeutic target for cancer
    It’s known to play important roles in tumor cell proliferation and survival, and it’s been shown to be an important mediator of resistance to targeted therapies such as EGFR inhibitors and conventional cytotoxic agents.
    mTOR/Raptor drives a feedback loop, mediated via IRS-1 phosphorylation that normally keeps PI3K activity in check.
    One consequence of mTOR/raptor inhibition by rapamycin and rapamycin analogs is alleviation of this negative feedback loop resulting in activation of PI3K and subsequent activation of AKT.
    Therefore, simultaneously targeting both PI3K and mTOR has the potential to simultaneously inhibit both upstream and downstream signaling in the pathway.
  • PI3K pathway is an exciting and promising therapeutic target for cancer
    It’s known to play important roles in tumor cell proliferation and survival, and it’s been shown to be an important mediator of resistance to targeted therapies such as EGFR inhibitors and conventional cytotoxic agents.
    mTOR/Raptor drives a feedback loop, mediated via IRS-1 phosphorylation that normally keeps PI3K activity in check.
    One consequence of mTOR/raptor inhibition by rapamycin and rapamycin analogs is alleviation of this negative feedback loop resulting in activation of PI3K and subsequent activation of AKT.
    Therefore, simultaneously targeting both PI3K and mTOR has the potential to simultaneously inhibit both upstream and downstream signaling in the pathway.
  • Based on this biology outlined in last slide, XL765 was rationally developed as a dual inhibitor of both PI3K and mTOR.
    As you can see XL765 inhibits Class I PI3Ks and mTOR and is highly specific for these targets over a large panel of other kinases.
  • PI3K pathway is an exciting and promising therapeutic target for cancer
    It’s known to play important roles in tumor cell proliferation and survival, and it’s been shown to be an important mediator of resistance to targeted therapies such as EGFR inhibitors and conventional cytotoxic agents.
    mTOR/Raptor drives a feedback loop, mediated via IRS-1 phosphorylation that normally keeps PI3K activity in check.
    One consequence of mTOR/raptor inhibition by rapamycin and rapamycin analogs is alleviation of this negative feedback loop resulting in activation of PI3K and subsequent activation of AKT.
    Therefore, simultaneously targeting both PI3K and mTOR has the potential to simultaneously inhibit both upstream and downstream signaling in the pathway.
  • PPT

    1. 1. The Future of Glioblastoma Therapy: Multi-modality with Multiple Targets Gautam Prasad Resident Physician Grand Rounds: May 15, 2009
    2. 2. Outline • Case Presentation • Moving Beyond Local Therapy • Potential Molecular Targets for GBM and a Case Example • XL765 – a dual PI3K/mTOR inhibitor • Preclinical Data with XL765 • Model Systems • In vitro – cytotoxicity and downstream molecular changes • In vivo – survival and disease burden in mice • Clinical Data with XL765 • Future Directions
    3. 3. Case Presentation • Pt J.E. is a 33M RH physician • Initial presentation and work-up • 1/09 – began experiencing worsening L frontal HA; pt reports awakening at night w/ pain accompanied by N/V • 2/9/09 – CT Head: 8.8 x 5.9 cm R frontal lobe mass w/ mass effect and R  L shift • 3/3/08 – Pre-operative MRI 4/6/08 – Post-op MRI - GTR
    4. 4. Case Presentation • Standard treatment (Stupp, NEJM 2005): 60 Gy + 75 mg/m2 of TMZ  4 week break  6 additional cycles of TMZ Median Survival: 14.6 months (12.1 months control) Median Surival w/ Methylated MGMT promoter: 21.7 months (15.3 months control) No Clinical Trials Available! Given age and KPS pt has < 4 years survival in all likelihood.
    5. 5. Outline • Case Presentation • Moving Beyond Local Therapy • Potential Molecular Targets for GBM and a Case Example • XL765 – a dual PI3K/mTOR inhibitor • Preclinical Data with XL765 • Model Systems • In vitro – cytotoxicity and downstream molecular changes • In vivo – survival and disease burden in mice • Clinical Data with XL765 • Future Directions
    6. 6. Local Therapy Alone is not the Solution “Isolation and characterization of human malignant glioma cells from histologically normal brain” Department of Neurosurgery, Washington University (J Neurosurgery 1997) 3 adults with supratentorial GBMs had resections in addition to biopsies of “normal” brain ≥ 4 cm from tumor * Normal brain biopsy
    7. 7. Local Therapy Alone is not the Solution Gross Tumor “Normal” Brain Cultured Glia Tumor cells? Yes No No GFAP + + + Growth Rate 19%/day 36%/day ~5%/day Motility 3.92 4.18 Karyotype Neoplastic Neoplastic Normal
    8. 8. Local Therapy Alone is not the Solution Conclusions Obviously a limited study (n = 3), but several interesting findings: 1. Histopathologic examination of frozen sections to determine what constitutes “normal” brain may be misleading 2. “Normal” brain in GBM patients may be infiltrated by tumor cells (?stem cells) as evidence by examination in culture 3. Focusing on the gross tumor (+ margin) alone will probably prevent death by herniation in the short-term but is not very what about increasing long-term survival?
    9. 9. Outline • Case Presentation • Moving Beyond Local Therapy • Potential Molecular Targets for GBM and a Case Example • XL765: a dual PI3K/mTOR inhibitor • Preclinical Data with XL765 • Model Systems • In vitro – cytotoxicity and downstream molecular changes • In vivo – survival and disease burden in mice • Clinical Data with XL765 • Future Directions
    10. 10. GBM – No Shortage of Potential Targets Source: Argyriou AA and Kalofonos HP 2009, Mol Med
    11. 11. PIP2 PI3K PIP3 EGFR PTEN IRS1 SurvivalRictor mTOR GßL 4EBP1 PRAS40 Raptor p70S6K Cell growth mTOR AKT GßL PI3K Signaling Pathway EGFEGF
    12. 12. PI3K Signaling Problems in GBMs 1. EGFR amplified (~40%) 2. EGFR overexpressed (~60%) 3. LOH 10q (~70%) 4. PI3K mutated/amplified (~20%) Source: Redmond KJ and Kleinberg LR 2009, Principles & Practice of Oncology
    13. 13. GBM – Targeting mTOR (Clinical Trial) Antitumor Activity of Rapamycin in a Phase I Trial for Patients with Recurrent PTEN-Deficient Glioblastoma Tim F. Cloughesy, Koji Yoshimoto, Phioanh Nghiemphu, et. al. PLoS Medicine, Jan 2008 165 pts in original cohort 14 PTEN deficient pts selected
    14. 14. GBM – Targeting mTOR (Clinical Trial) After one week of treatment with Rapamycin: 1. 7 of 14 (50%) of pts had a substantial reduction in mTOR levels which coorelated well with tumor proliferation (p = 0.005) 2. Tumor cells harvested from non-responders did respond to rapamycin ex vivo. Therefore there was nothing intrinsic in the cells themselves that caused resistance. 3. 7 of 14 (50%) of pts had up-regulation of Akt (loss of negative feedback) which led to shortened time-to-progression (p = 0.05). Conclusion: There is value in inhibition of the Akt/PI3K pathway through mTOR but as the TTP curves show, combination with a second inhibitor (e.g. PI3K or EGFR) would be valuable.
    15. 15. PIP2 PI3K PIP3 EGFR PTEN IRS1 SurvivalRictor mTOR GßL 4EBP1 PRAS40 Raptor p70S6K Cell growth mTOR AKT GßL PI3K Signaling Pathway EGFEGF Resistance to Rapamycin via p70S6K:IRS pathway Rapa Analogs
    16. 16. Outline • Case Presentation • Moving Beyond Local Therapy • Potential Molecular Targets for GBM and a Case Example • XL765: a dual PI3K/mTOR inhibitor • Preclinical Data with XL765 • Model Systems • In vitro – cytotoxicity and downstream molecular changes • In vivo – survival and disease burden in mice • Clinical Data with XL765 • Future Directions
    17. 17. Family Kinase IC50 (nM) PI3K Class IA PI3Kα 39 PI3Kβ 113 PI3Kδ 43 Class IB PI3Kγ 9 Class III VPS34 9000 PIKK (PI3K-related) DNA-PK 150 mTOR 157 XL765: A Potent PI3K/mTOR Inhibitor Inhibition of Class I PI3K isoforms and mTOR ATP competitive and reversible binding Highly selective in panel of > 120 kinases p70S6K 4EBP1 PRAS40 mTOR AKTmTOR PI3K Raptor Rictor S6 XL765 XL765 XL765
    18. 18. GBM Xenografts: Clinical and Biological Data Xenograft Clinical Information EGFR PTEN GBM6 65M, Frontal, OS 13 mo VIII wt GBM8 74F, Frontal, OS 16 mo wt null GBM12 68M, Occiptal, OS 3 mo* wt wt GBM GS-2 57M, Occipital, 2nd resection wt null GBM 39 51M, Frontal, OS 20 mo VIII wt * Pt died of pulmonary embolus (NED at time)
    19. 19. 0% 20% 40% 60% 80% 100% 120% Negative 0.5 1.0 2.0 4.0 8.0 12.0 16.0 20.0XL 765 (in µM) RelativeCellViability 0% 20% 40% 60% 80% 100% 120% N egative 0.5 1.0 2.0 4.0 8.0 12.0 16.0 20.0 XL 765 (in µM) RelativeCellViability 0% 20% 40% 60% 80% 100% 120% Negative 0.5 1.0 2.0 4.0 8.0 12.0 20.0 XL 765 (in µM) RelativeCellViability 0% 20% 40% 60% 80% 100% 120% N egative 0.5 1.0 2.0 4.0 8.0 12.0 16.0 20.0 XL 765 (in µM) RelativeCellViability GBM 6 (EGFR VIII, PTEN wt) IC50 = 7.5 µM In vitro - XL 765 Effects on Cell Viability GBM 8 (EGFR wt, PTEN null) IC50 = 4.0 µM GBM 12 (EGFR wt, PTEN wt) IC50 = 2.0 µM GBM GS-2 (EGFR wt, PTEN null) IC50 = 4.0 µM
    20. 20. Control XL 765 (µM) 1 2 4 8 16 pAktser473 pPRAS40thr246 pS6ser235/236 p4EBP1thr37/46 Actin GBM 6 GBM GBM12 GBM GS-2 EGFR VIII, PTEN wt EGFR wt, PTEN null EGFR wt, PTEN wt EGFR wt, PTEN null Control XL 765 (µM) 1 2 4 8 16 Control XL 765 (µM) 1 2 4 8 16 Control XL 765 (µM) 1 2 4 8 16 In vitro - Downstream Changes in the PI3K Pathway
    21. 21. In vitro - XL 765 + TMZ + XRT on Cell Viability GBM12 (EGFR wt, PTEN wt) GBM6 (EGFR VIII, PTEN wt) GBM8 (EGFR wt, PTEN null) GBM GS2 (EGFR wt, PTEN null) 0 20 40 60 80 100 120 Control XL 765 2.5uM XRT 8 Gy Tem odar800 uM 765+ XRT Tem odar+ XRT 765+ Tem odar All3 RelativeCellViability * * * * * * 0 20 40 60 80 100 120 ControlXL 765 8 uM XRT 8 Gy Tem odar800 uM 765+ XRT Tem odar+ XRT 765+ Tem odar All3 RelativeCellViability * * * 0 20 40 60 80 100 120 Control XL 765 2.5uM XRT 8 Gy Tem odar800 uM 765+ XRT Tem odar+ XRT765+ Tem odar All3 RelativeCellViability * * * * * * p < 0.05 0 20 40 60 80 100 120 ControlXL7654.0uM XRT8Gy Temodar800uM 765+XRT Temodar+XRT765+Temodar All3 RelativeCellViability * * * * *
    22. 22. PIP2 PI3K PIP3 EGFR PTEN IRS1 SurvivalRictor mTOR GßL 4EBP1 PRAS40 Raptor p70S6K Cell growth mTOR AKT GßL PI3K Signaling Pathway EGFEGF XL765 XL765 XL765
    23. 23. Nude mouse with serially passaged subcutaneous xenograft Xenograft removed and diced Intracranial injection of xenograft In vivo - Methodology
    24. 24. In vivo - Methodology Agent Route Control Oral gavage w/ Ora-Care Plus XL 765 (XL) Oral gavage w/ XL dissolved in sterile saline TMZ Oral gavage w/ TMZ dissolved in Ora-Care Plus Erlotinib (ERL) Oral gavage w/ ERL dissolved in sterile saline XRT Single lateral Cs-137 beam through head; body shielded w/ Pb
    25. 25. Intracranial injection of xenograft In vivo Methodology Day 1 5-20 Mice optically imaged and sorted into groups of 10 20-30 Mice treated with XL765 bid and/or TMZ qd by oral gavage 50-60 Repeat treatments ** Mice optically imaged 3/week during first 2 months and weighed daily during treatment
    26. 26. In vivo – Control vs XRT Days s/p implantation AverageRadiance Click # SM20081006115606 Mon, Oct 06, 2008 11:56:18 Bin:M (8), FOV25, f1, 30s Filter: Open Camera: IVIS 13040, SI620EEV Series Exper Label Comm Analy ROI 1=1.1075e+08ROI 2=4.5634e+07ROI 3=3.4216e+06RO Total: Area Flux = 1.97694e+08 Day 18 Control XRT Click # SM20081006122331 Mon, Oct 06, 2008 12:23:45 Bin:M (8), FOV25, f1, 30s Filter: Open Camera: IVIS 13040, SI620EEV Series: Experiment: GBM12 Label: 388, 86, 393, 100 Comment: Analysis Comment: ROI 1=1.3218e+07 ROI 2=1.9004e+07 ROI 3=3.1335e+07 ROI 4=5.5907e+06 Total: Area Flux = 6.91471e+07 Tx #1 GBM 12 (EGFR wt, PTEN wt) p = 0.15
    27. 27. Tx #1 Tx #2 Days s/p implantation AverageRadianceIn vivo – XL765 ± TMZ GBM 39 (EGFR VIII, PTEN wt) MGMT hyper-methylated Day 46 Click # SM20090320121757 Fri, Mar 20, 2009 12:18:10 Bin:M (8), FOV25, f1, 5s Filter: Open Camera: IVIS 13040, SI620EEV Series: Experiment: GBM39 Label: Comment: Analysis Comment: 100 80 60 40 20 Imag Min = -1.72 Max = 1.66 p/sec/cm Color Min = 5.96 Max = 1.19 bkg sub flat-fielded cosmic WARNING: Saturated Luminescent Image ROI 1=9.0168e+08 ROI 2=2.454e+08 ROI 3=2.9723e+07 ROI 4=1.3079e+09 Total: Area Flux = 2.48469e+09 Click # SM20090320122520 Fri, Mar 20, 2009 12:25:33 Bin:M (8), FOV25, f1, 5s Filter: Open Camera: IVIS 13040, SI620EEV Series: Experiment: G Label: Comment: Analysis Com ROI 1=4.3235e+06 ROI 2=2.7163e+08 ROI 3=1.0863e+08 ROI 4=1.47 Total: Area Flux = 6.01055e+08 Control XL p = 0.001 ROI 1=3.3586e+06 ROI 2=3.3204e+07 ROI 3=6.2986e+05 R Total: Area Flux = 6.5042e+07 ROI 1=2.7215e+05 ROI 2=7.3402e Total: Area Flux = 1.64832e+07 TMZ XL+TMZ p = 0.0002 p = 0.063
    28. 28. In vivo – XL ± ERL Tx #1 Tx #2 Days s/p implantation AverageRadiance 10 8 6 4 2 x10 6 Image Min = -1.6611e+09 Max = 1.3777e+08 p/sec/cm^2/sr Color Bar Min = 1.1835e+05 Max = 1.0558e+07 bkg sub flat-fielded ROI 1=5.8971e+05 ROI 2=6.8719e+06 ROI 3=4.6397e+07 ROI 4=2.7683e+06 ROI 5=4.057e+06 Total: Area Flux = 6.06837e+07 Click # SM20090320124719 Fri, Mar 20, 2009 12:47:32 Series: Experiment: GBM39 50 40 30 20 10 Im Min = -3. Max = 5. p/sec/ Colo Min = 2. Max = 5. bkg sub flat-fielde cosmic ROI 1=3.3164e+08 ROI 2=2.9054e+06 ROI 3=7.7691e+07 ROI 4=2.6526e+07 ROI 5=1.4211e+07 Total: Area Flux = 4.52977e+08 GBM 39 (EGFR VIII, PTEN wt) MGMT hyper-methylated Day 46 Click # SM20090320121757 Fri, Mar 20, 2009 12:18:10 Bin:M (8), FOV25, f1, 5s Filter: Open Camera: IVIS 13040, SI620EEV Series: Experiment: GBM39 Label: Comment: Analysis Comment: 100 80 60 40 20 Imag Min = -1.72 Max = 1.66 p/sec/cm Color Min = 5.96 Max = 1.19 bkg sub flat-fielded cosmic WARNING: Saturated Luminescent Image ROI 1=9.0168e+08 ROI 2=2.454e+08 ROI 3=2.9723e+07 ROI 4=1.3079e+09 Total: Area Flux = 2.48469e+09 Click # SM20090320122520 Fri, Mar 20, 2009 12:25:33 Bin:M (8), FOV25, f1, 5s Filter: Open Camera: IVIS 13040, SI620EEV Series: Experiment: G Label: Comment: Analysis Com ROI 1=4.3235e+06 ROI 2=2.7163e+08 ROI 3=1.0863e+08 ROI 4=1.47 Total: Area Flux = 6.01055e+08 Control XL p = 0.001 ERL XL+ERL p < 0.0001 p = 0.97
    29. 29. Group Median Survival Control 25 XRT 32 In vivo Survival – Control vs. XRT Group p HR XRT vs Control 0.001 13.3 (2.7-65.3) 20 25 30 35 40 100 80 60 40 20 0 Time (Days) Survivalprobability(%) Control XRT
    30. 30. 40 50 60 70 80 90 100 80 60 40 20 0 Time (Days) Survivalprobability(%) Control TMZ XL XL+TMZ Group Median Survival Control 54.5 XL 67.5 TMZ 82.5 XL+TMZ N/R In vivo Survival – XL ± TMZ Groups p HR XL vs Control 0.06 2.8 (1.0-7.8) TMZ vs Control 0.0001 12.4 (3.5-43.9) XL+TMZ vs TMZ 0.08 4.6 (1.1-19.4)
    31. 31. 40 50 60 70 80 90 100 80 60 40 20 0 Time (Days) Survivalprobability(%) Control Erl XL XL+Erl In vivo Survival – XL ± ERL Group Median Survival Control 54.5 XL 67.5 ERL 77 XL+ERL 78.5 Group p HR XL vs Control 0.06 2.8 (1.0-7.8) ERL vs Control 0.0002 11.0 (3.1-38.9) XL+ERL vs ERL 0.44 0.6 (0.2-1.6)
    32. 32. Preclinical Data Summary In Vitro • XL765 results in concentration-dependent cytotoxicity alone and is supra-additive when combined with conventional agents. •In addition, the PI3K/mTOR pathway is specifically inhibited as demonstrated by Western Blot. In Vivo •XL765 given as monotherapy in mice with intracranial GBM xenografts resulted in improved survival. •Combination of XL765 with TMZ resulted in a trend for decreased tumor growth and survival. •Combination of XL765 with Erlotinib did not demonstrate any additive effects in the model we tested.
    33. 33. Outline • Case Presentation • Moving Beyond Local Therapy • Potential Molecular Targets for GBM and a Case Example • XL765 – a dual PI3K/mTOR inhibitor • Preclinical Data with XL765 • Model Systems • In vitro – cytotoxicity and downstream molecular changes • In vivo – survival and disease burden in mice • Clinical Data with XL765 • Future Directions
    34. 34. Clinical Data in Humans Phase I dose-escalation study Presented at the joint EORTC-NCI-AACR conference in Geneva 10/08 Vall d’Hebron Hospital (Barcelona, Spain), Karmanos Cancer Center (Detroit, MI), START Medical Oncology (San Antonio, TX) 1. 29 patients w/ metastatic or unresectable solid tumor for which no further effective measures exist 2. No chemotherapy, radiotherapy, or biological agents within 30 days 3. Primary objective: saftey and tolerability 4. Secondary objectives: PK/PD/preliminary efficacy
    35. 35. Clinical Data – Doses & Status
    36. 36. Clinical Data - Toxicity
    37. 37. Clinical Data - Response Note decrease in phospo- Akt and phospho-4EBP1 in patient hair follicles after treatment
    38. 38. Clinical Data - Conclusions 1. XL765 was generally well-tolerated w/ GI complaints being most common; no MTD reached 2. XL765 showed pharmacodynamic response in hair follicles, skin cells, and in cases of tumor biopsy. Phase I dose-escalation study of XL765 + TMZ in adults w/ malignant gliomas University of California Los Angeles and Memorial Sloan-Kettering Cancer Center • Patients need to be on Temozlomide already at a dose of 200 mg/m2/day on days 1-5 of 28 day cycle • Patients must have completed four cycles w/o unacceptable toxicity • NO progression on temozlomide • Currently accruing
    39. 39. Future Directions 1. IHC examination of treated in vivo xenografts 2. In vivo model using XL 765 + Erlotinib 3. Clinical Trial @ UCSF • Phase I • Fixed dose XL 765 + escalating Erlotinib doses
    40. 40. Haas-Kogan Lab
    41. 41. Haas-Kogan Lab (the reality)
    42. 42. Acknowledgements Daphne Haas-Kogan Michael Prados Theo Sottero Xiaodong Yang Sabine Mueller C. David James Mei-Yin Polley Tomoko Ozawa Raquel Santos Dana Aftab

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