Robert Lemos

Mutations in the phosphatidylinositol-3-kinase pathway predict for antitumor activity of the inhibitor PX-866 whereas oncogenic ras is a dominant predictor for resistance

The novel phosphatidylinositol-3-kinase (PI3K) inhibitor PX-866 was tested against 13 experimental human tumor xenografts derived from cell lines of various tissue origins. Mutant PI3K (PIK3CA) and loss of PTEN activity were sufficient, but not necessary, as predictors of sensitivity to the antitumor activity of the PI3K inhibitor PX-866 in the presence of wild-type Ras, whereas mutant oncogenic Ras was a dominant determinant of resistance, even in tumors with coexisting mutations in PIK3CA. The level of activation of PI3K signaling measured by tumor phosphorylated Ser(473)-Akt was insufficient to predict in vivo antitumor response to PX-866. Reverse-phase protein array revealed that the Ras-dependent downstream targets c-Myc and cyclin B were elevated in cell lines resistant to PX-866 in vivo. Studies using an H-Ras construct to constitutively and preferentially activate the three best-defined downstream targets of Ras, i.e., Raf, RalGDS, and PI3K, showed that mutant Ras mediates resistance through its ability to use multiple pathways for tumorigenesis. The identification of Ras and downstream signaling pathways driving resistance to PI3K inhibition might serve as an important guide for patient selection as inhibitors enter clinical trials and for the development of rational combinations with other molecularly targeted agents.

Resistance to BRAF Inhibition in BRAF-Mutant Colon Cancer Can Be Overcome with PI3K Inhibition or Demethylating Agents

Purpose: Vemurafenib, a selective inhibitor of BRAFV600, has shown significant activity in BRAFV600 melanoma but not in less than 10% of metastatic BRAFV600 colorectal cancers (CRC), suggesting that studies of the unique hypermethylated phenotype and concurrent oncogenic activation of BRAFmut CRC may provide combinatorial strategies. Experimental Design: We conducted comparative proteomic analysis of BRAFV600E melanoma and CRC cell lines, followed by correlation of phosphoinositide 3-kinase (PI3K) pathway activation and sensitivity to the vemurafenib analogue PLX4720. Pharmacologic inhibitors and siRNA were used in combination with PLX4720 to inhibit PI3K and methyltransferase in cell lines and murine models. Results: Compared with melanoma, CRC lines show higher levels of PI3K/AKT pathway activation. CRC cell lines with mutations in PTEN or PIK3CA were less sensitive to growth inhibition by PLX4720 (P = 0.03), and knockdown of PTEN expression in sensitive CRC cells reduced growth inhibition by the drug. Combined treatment of PLX4720 with PI3K inhibitors caused synergistic growth inhibition in BRAF-mutant CRC cells with both primary and secondary resistance. In addition, methyltransferase inhibition was synergistic with PLX4720 and decreased AKT activation. In vivo, PLX4720 combined with either inhibitors of AKT or methyltransferase showed greater tumor growth inhibition than PLX4720 alone. Clones with acquired resistance to PLX4720 in vitro showed PI3K/AKT activation with EGF receptor (EGFR) or KRAS amplification. Conclusions: We show that activation of the PI3K/AKT pathway is a mechanism of both innate and acquired resistance to BRAF inhibitors in BRAFV600E CRC and suggest combinatorial approaches to improve outcomes in this poor prognosis subset of patients. Clin Cancer Res; 19(3); 657–67. ©2012 AACR.

The hypoxia-associated factor switches cells from HIF-1α- to HIF-2α-dependent signaling promoting stem cell characteristics, aggressive tumor growth and invasion.

Most solid tumors and their metastases experience periods of low oxygen or hypoxia, which is of major clinical significance as it promotes both tumor progression and resistance to therapy. Critical mediators of the hypoxic response are the hypoxia-inducible factors HIF-1α and HIF-2α. The HIFs are nonredundant and regulate both overlapping and unique downstream target genes. Here, we describe a novel mechanism for the switch between HIF-1α- and HIF-2α-dependent transcription during tumor hypoxia caused by the hypoxia associated factor (HAF). HAF is overexpressed in a variety of tumors and its levels are decreased during acute hypoxia, but increased following prolonged hypoxia. We have previously identified HAF as an E3 ubiquitin ligase that binds and ubiquitinates HIF-1α by an oxygen and pVHL-independent mechanism, thus targeting HIF-1α for proteasomal degradation. Here, we show that HAF also binds to HIF-2α, but at a different site than HIF-1α, and increases HIF-2α transactivation without causing its degradation. HAF, thus, switches the hypoxic response of the cancer cell from HIF-1α-dependent to HIF-2α-dependent transcription and activates genes involved in invasion such as MMP9, PAI-1, and the stem cell factor OCT-3/4. The switch to HIF-2α-dependent gene expression caused by HAF also promotes an enriched tumor stem cell population, resulting in highly aggressive tumors in vivo. Thus, HAF, by causing a switch from a HIF-1α- to HIF-2α-dependent response to hypoxia, provides a mechanism for more aggressive growth of tumors under prolonged hypoxia.

The Promise of Patient-Derived Xenografts: The Best Laid Plans of Mice and Men

Compared with xenografts from previously established cell lines, patient-derived xenografts may more faithfully recapitulate the molecular diversity, cellular heterogeneity, and histology seen in patient tumors, although other limitations of murine models remain. The ability of these models to inform clinical development and answer mechanistic questions will determine their ultimate use. Clin Cancer Res; 18(19); 5160–2. ©2012 AACR.

The selective hypoxia inducible factor-1 inhibitor PX-478 provides in vivo radiosensitization through tumor stromal effects.

Hypoxia inducible factor-1 (HIF-1) promotes tumor cell adaptation to microenvironmental stress. HIF-1 is up-regulated in irradiated tumors and serves as a promising target for radiosensitization. We initially confirmed that the orally bioavailable HIF-1 inhibitor PX-478 reduces HIF-1 protein levels and signaling in vitro in a dose-dependent manner and provides direct radiosensitization of hypoxic cancer cells in clonogenic survival assays using C6 glioma, HN5 and UMSCCa10 squamous cells, and Panc-1 pancreatic adenocarcinoma cell lines. However, PX-478 yields striking in vivo tumor sensitization to single-dose irradiation, which cannot be explained by incremental improvement in direct tumor cell killing. We show that PX-478 prevents postradiation HIF-1 signaling and abrogates downstream stromal adaptation in C6 and HN5 reporter xenografts as measured by serial ultrasound, vascular magnetic resonance imaging, and hypoxia response element–specific micro–positron emission tomography imaging. The primacy of indirect PX-478 in vivo effects was corroborated by our findings that (a) either concurrent or early postradiation sequencing of PX-478 provides roughly equivalent sensitization and (b) constitutive vascular endothelial growth factor expression maintains refractory tumor vessel function and progression following combined radiation and PX-478. These results confirm that disruption of postradiation adaptive HIF-1 signaling by PX-478 imparts increased therapeutic efficacy through blockade of HIF-1–dependent reconstitution of tumor stromal function. Successful translation of targeted HIF-1 radiosensitization to the clinical setting will require specific consideration of tumor microenvironmental effects and mechanisms. [Mol Cancer Ther 2009;8(4):947–58]

Radiosensitization and Stromal Imaging Response Correlates for the HIF-1 Inhibitor PX-478 Given with or without Chemotherapy in Pancreatic Cancer

Growing tumors are hypoxic and respond to microenvironmental stress through increased expression of the hypoxia inducible factor-1α (HIF-1α) transcription factor, resulting in an adaptive switch to glycolytic metabolism, angiogenic signaling, survival, and metastasis. HIF-1α expression is associated with tumor resistance to cytotoxic therapy and inferior patient outcomes. Pancreatic cancer is the most hypoxic of all solid tumors and remains refractory to current chemoradiotherapy. We have seen nuclear HIF-1α in 88% of human pancreatic ductal carcinoma but in only 16% of normal pancreas. Stroma adjacent to the pancreatic ductal carcinoma also showed HIF-1α in 43% of cases. We investigated the novel selective HIF-1α inhibitor PX-478 on in vitro and in vivo radiation response of human pancreatic cancer models. Inhibition of HIF-1α by PX-478 increased cell killing by radiation. In mice with Panc-1, CF-PAC-1, or SU.86.86 pancreatic xenografts, concurrent administration of PX-478 potentiated the antitumor effects of fractionated radiation, with or without combined treatment with 5-fluorouracil or gemcitabine. Alternative sequencing of PX-478 with fractionated radiotherapy suggests optimal radiosensitization with concurrent or neoadjuvant administration of drug. Early tumor responses to combined PX-478/radiation treatment could be rapidly and repeatedly quantified by vascular imaging biomarkers. Dual-tracer dynamic contrast enhanced–magnetic resonance imaging and ultrasound imaging discriminated response to combined treatment prior to detection of differences in anatomic tumor size at 10 days posttreatment. Therefore, PX-478 is a mechanistically appealing and potentially clinically relevant enhancer of pancreatic cancer radiosensitivity, inhibiting tumor and stromal HIF-1 proangiogenic signaling and reducing the innate radiation resistance of hypoxic tumor cells. Mol Cancer Ther; 9(7); 2057–67. ©2010 AACR.

Molecular Pharmacology and Antitumor Activity of PHT-427, a Novel Akt/Phosphatidylinositide-Dependent Protein Kinase 1 Pleckstrin Homology Domain Inhibitor

Phosphatidylinositol 3-kinase/phosphatidylinositide-dependent protein kinase 1 (PDPK1)/Akt signaling plays a critical role in activating proliferation and survival pathways within cancer cells. We report the molecular pharmacology and antitumor activity of PHT-427, a compound designed to bind to the pleckstrin homology (PH) binding domain of signaling molecules important in cancer. Although originally designed to bind the PH domain of Akt, we now report that PHT-427 also binds to the PH domain of PDPK1. A series of PHT-427 analogues with variable C-4 to C-16 alkyl chain length were synthesized and tested. PHT-427 itself (C-12 chain) bound with the highest affinity to the PH domains of both PDPK1 and Akt. PHT-427 inhibited Akt and PDPK1 signaling and their downstream targets in sensitive but not resistant cells and tumor xenografts. When given orally, PHT-427 inhibited the growth of human tumor xenografts in immunodeficient mice, with up to 80% inhibition in the most sensitive tumors, and showed greater activity than analogues with C4, C6, or C8 alkyl chains. Inhibition of PDPK1 was more closely correlated to antitumor activity than Akt inhibition. Tumors with PIK3CA mutation were the most sensitive, and K-Ras mutant tumors were the least sensitive. Combination studies showed that PHT-427 has greater than additive antitumor activity with paclitaxel in breast cancer and with erlotinib in non–small cell lung cancer. When given >5 days, PHT-427 caused no weight loss or change in blood chemistry. Thus, we report a novel PH domain binding inhibitor of PDPK1/Akt signaling with significant in vivo antitumor activity and minimal toxicity. Mol Cancer Ther; 9(3); 706–17

In vitro and In vivo Activity of Novel Small-Molecule Inhibitors Targeting the Pleckstrin Homology Domain of Protein Kinase B/AKT

The phosphatidylinositol 3-kinase/AKT signaling pathway plays a critical role in activating survival and antiapoptotic pathways within cancer cells. Several studies have shown that this pathway is constitutively activated in many different cancer types. The goal of this study was to discover novel compounds that bind to the pleckstrin homology (PH) domain of AKT, thereby inhibiting AKT activation. Using proprietary docking software, 22 potential PH domain inhibitors were identified. Surface plasmon resonance spectroscopy was used to measure the binding of the compounds to the expressed PH domain of AKT followed by an in vitro activity screen in Panc-1 and MiaPaCa-2 pancreatic cancer cell lines. We identified a novel chemical scaffold in several of the compounds that binds selectively to the PH domain of AKT, inducing a decrease in AKT activation and causing apoptosis at low micromolar concentrations. Structural modifications of the scaffold led to compounds with enhanced inhibitory activity in cells. One compound, 4-dodecyl-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide, inhibited AKT and its downstream targets in cells as well as in pancreatic cancer cell xenografts in immunocompromised mice; it also exhibited good antitumor activity. In summary, a pharmacophore for PH domain inhibitors targeting AKT function was developed. Computer-aided modeling, synthesis, and testing produced novel AKT PH domain inhibitors that exhibit promising preclinical properties.

Peroxisome proliferator-activated receptor γ agonist pioglitazone prevents the hyperglycemia caused by phosphatidylinositol 3-kinase pathway inhibition by PX-866 without affecting antitumor activity

The phosphatidylinositol 3-kinase (PI3K)/Akt signaling cascade is an important component of the insulin signaling in normal tissues leading to glucose uptake and homeostasis and for cell survival signaling in cancer cells. Hyperglycemia is an on-target side effect of many inhibitors of PI3K/Akt signaling including the specific PI3K inhibitor PX-866. The peroxisome proliferator-activated receptor γ agonist pioglitazone, used to treat type 2 diabetes, prevents a decrease in glucose tolerance caused by acute administration of PX-866. Our studies have shown that pioglitazone does not inhibit the antitumor activity of PX-866 in A-549 non-small cell lung cancer and HT-29 colon cancer xenografts. In vitro studies also showed that pioglitazone increases 2-[1-14C]deoxy-d-glucose uptake in L-6 muscle cells and prevents inhibition of 2-deoxyglucose uptake by PX-866. Neither pioglitazone nor PX-866 had an effect on 2-deoxyglucose uptake in A-549 lung cancer cells. In vivo imaging studies using [18F]2-deoxyglucose (FDG) positron emission tomography showed that pioglitazone increases FDG accumulation by normal tissue but does not significantly alter FDG uptake by A-549 xenografts. Thus, peroxisome proliferator-activated receptor γ agonists may be useful in overcoming the increase in blood glucose caused by inhibitors of PI3K signaling by preventing the inhibition of normal tissue insulin-mediated glucose uptake without affecting antitumor activity. [Mol Cancer Ther 2009;8(1):94–100]

HIF-1–Dependent Stromal Adaptation to Ischemia Mediates In Vivo Tumor Radiation Resistance

Purpose: Hypoxia-inducible factor 1 (HIF-1) promotes cancer cell survival and tumor progression. The specific role played by HIF-1 and tumor–stromal interactions toward determining tumor resistance to radiation treatment remains undefined. We applied a multimodality preclinical imaging platform to mechanistically characterize tumor response to radiation, with a focus on HIF-1–dependent resistance pathways. Methods: C6 glioma and HN5 human squamous carcinoma cells were stably transfected with a dual HIF-1 signaling reporter construct (dxHRE-tk/eGFP-cmvRed2XPRT). Reporter cells were serially interrogated in vitro before and after irradiation as monolayer and multicellular spheroid cultures and as subcutaneous xenografts in nu/nu mice. Results: In vitro, single-dose irradiation of C6 and HN5 reporter cells modestly impacted HIF-1 signaling in normoxic monolayers and inhibited HIF-1 signaling in maturing spheroids. In contrast, irradiation of C6 or HN5 reporter xenografts with 8 Gy in vivo elicited marked upregulation of HIF-1 signaling and downstream proangiogenic signaling at 48 hours which preceded recovery of tumor growth. In situ ultrasound imaging and dynamic contrast-enhanced (DCE) MRI indicated that HIF-1 signaling followed acute disruption of stromal vascular function. High-resolution positron emission tomography and dual-contrast DCE-MRI of immobilized dorsal skin window tumors confirmed postradiotherapy HIF-1 signaling to spatiotemporally coincide with impaired stromal vascular function. Targeted disruption of HIF-1 signaling established this pathway to be a determinant of tumor radioresistance. Conclusions: Our results illustrate that tumor radioresistance is mediated by a capacity to compensate for stromal vascular disruption through HIF-1–dependent proangiogenic signaling and that clinically relevant vascular imaging techniques can spatially define mechanisms associated with tumor irradiation. Mol Cancer Res; 9(3); 259–70. ©2011 AACR.