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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 PPT Presentation Transcript

  • The Future of Glioblastoma Therapy: Multi-modality with Multiple Targets Gautam Prasad Resident Physician Grand Rounds: May 15, 2009
    • 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
    Outline
    • 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
    Case Presentation
    • Standard treatment (Stupp, NEJM 2005):
    • 60 Gy + 75 mg/m 2 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. Case Presentation
    • 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
    Outline
  • “ 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 Local Therapy Alone is not the Solution
  • 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
    • Conclusions
    • Obviously a limited study (n = 3), but several interesting findings:
    • Histopathologic examination of frozen sections to determine what constitutes “normal” brain may be misleading
    • “ Normal” brain in GBM patients may be infiltrated by tumor cells (?stem cells) as evidence by examination in culture
    • 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?
    Local Therapy Alone is not the Solution
    • 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
    Outline
  • Source: Argyriou AA and Kalofonos HP 2009, Mol Med GBM – No Shortage of Potential Targets
  • EGF PIP2 PI3K PIP3 EGFR PTEN IRS1 Survival Rictor mTOR GßL 4EBP1 PRAS40 Raptor p70S6K Cell growth mTOR AKT GßL PI3K Signaling Pathway
    • EGFR amplified (~40%)
    • EGFR overexpressed (~60%)
    • LOH 10q (~70%)
    • PI3K mutated/amplified (~20%)
    Source: Redmond KJ and Kleinberg LR 2009, Principles & Practice of Oncology PI3K Signaling Problems in GBMs
  • 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 GBM – Targeting mTOR (Clinical Trial)
    • After one week of treatment with Rapamycin:
    • 7 of 14 (50%) of pts had a substantial reduction in mTOR levels which coorelated well with tumor proliferation ( p = 0.005)
    • Tumor cells harvested from non-responders did respond to rapamycin ex vivo . Therefore there was nothing intrinsic in the cells themselves that caused resistance.
    • 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.
    GBM – Targeting mTOR (Clinical Trial)
  • EGF Resistance to Rapamycin via p70S6K:IRS pathway Rapa Analogs PIP2 PI3K PIP3 EGFR PTEN IRS1 Survival Rictor mTOR GßL 4EBP1 PRAS40 Raptor p70S6K Cell growth mTOR AKT GßL PI3K Signaling Pathway
    • 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
    Outline
  • 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 Family Kinase IC 50 (nM) PI3K Class IA PI3K  39 PI3K  113 PI3K  43 Class IB PI3K  9 Class III VPS34 9000 PIKK (PI3K-related) DNA-PK 150 mTOR 157 p70S6K 4EBP1 PRAS40 mTOR AKT mTOR PI3K Raptor Rictor S6 XL765 XL765 XL765
  • 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, 2 nd resection wt null GBM 39 51M, Frontal, OS 20 mo VIII wt * Pt died of pulmonary embolus (NED at time)
  • GBM 6 ( EGFR VIII, PTEN wt ) IC 50 = 7.5 µM In vitro - XL 765 Effects on Cell Viability GBM 8 ( EGFR wt, PTEN null ) IC 50 = 4.0 µM GBM 12 ( EGFR wt, PTEN wt ) IC 50 = 2.0 µM GBM GS-2 ( EGFR wt, PTEN null ) IC 50 = 4.0 µM
  • pAkt ser473 pPRAS40 thr246 pS6 ser235/236 p4EBP1 thr37/46 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 Control XL 765 ( µM) 1 2 4 8 16 In vitro - Downstream Changes in the PI3K Pathway
  • 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 ) * * * * * * * * * * * * * * * p < 0.05 * * * * *
  • EGF XL765 XL765 XL765 PIP2 PI3K PIP3 EGFR PTEN IRS1 Survival Rictor mTOR GßL 4EBP1 PRAS40 Raptor p70S6K Cell growth mTOR AKT GßL PI3K Signaling Pathway
  • Nude mouse with serially passaged subcutaneous xenograft Xenograft removed and diced Intracranial injection of xenograft In vivo - Methodology
  • 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
  • Intracranial injection of xenograft 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 In vivo Methodology
  • Days s/p implantation Average Radiance Day 18 Control XRT GBM 12 ( EGFR wt, PTEN wt ) p = 0.15 In vivo – Control vs XRT Tx #1
  • Days s/p implantation Average Radiance GBM 39 ( EGFR VIII, PTEN wt ) MGMT hyper-methylated Day 46 Tx #1 Tx #2 In vivo – XL765 ± TMZ Control XL p = 0.001 TMZ XL+TMZ p = 0.0002 p = 0.063
  • Days s/p implantation Average Radiance GBM 39 ( EGFR VIII, PTEN wt ) MGMT hyper-methylated Day 46 ERL XL+ERL p < 0.0001 p = 0.97 In vivo – XL ± ERL Tx #1 Tx #2 Control XL p = 0.001
  • 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)
  • 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)
  • 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)
    • 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.
    Preclinical Data Summary
    • 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
    Outline
    • 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)
    • 29 patients w/ metastatic or unresectable solid tumor for which no further effective measures exist
    • No chemotherapy, radiotherapy, or biological agents within 30 days
    • Primary objective: saftey and tolerability
    • Secondary objectives: PK/PD/preliminary efficacy
    Clinical Data in Humans
  • Clinical Data – Doses & Status
  • Clinical Data - Toxicity
  • Note decrease in phospo-Akt and phospho-4EBP1 in patient hair follicles after treatment Clinical Data - Response
    • XL765 was generally well-tolerated w/ GI complaints being most common; no MTD reached
    • 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
    Clinical Data - Conclusions
    • IHC examination of treated in vivo xenografts
    • In vivo model using XL 765 + Erlotinib
    • Clinical Trial @ UCSF
      • Phase I
      • Fixed dose XL 765 + escalating Erlotinib doses
    Future Directions
  • Haas-Kogan Lab
  • Haas-Kogan Lab (the reality)
      • Daphne Haas-Kogan Michael Prados
      • Theo Sottero
      • Xiaodong Yang
      • Sabine Mueller
      • C. David James Mei-Yin Polley
      • Tomoko Ozawa
      • Raquel Santos
      • Dana Aftab
    Acknowledgements