Jason D. Lickliter

EphA3 Maintains Tumorigenicity and Is a Therapeutic Target in Glioblastoma Multiforme

Significant endeavor has been applied to identify functional therapeutic targets in glioblastoma (GBM) to halt the growth of this aggressive cancer. We show that the receptor tyrosine kinase EphA3 is frequently overexpressed in GBM and, in particular, in the most aggressive mesenchymal subtype. Importantly, EphA3 is highly expressed on the tumor-initiating cell population in glioma and appears critically involved in maintaining tumor cells in a less differentiated state by modulating mitogen-activated protein kinase signaling. EphA3 knockdown or depletion of EphA3-positive tumor cells reduced tumorigenic potential to a degree comparable to treatment with a therapeutic radiolabelled EphA3-specific monoclonal antibody. These results identify EphA3 as a functional, targetable receptor in GBM.

Characterization of the Epha1 receptor tyrosine kinase: expression in epithelial tissues.

Abstract The Eph family of receptor tyrosine kinases plays a crucial role during development and is implicated in oncogenesis. Using a partial cDNA clone of an Eph-related kinase (Esk) we isolated the complete coding region of a gene which we show to be murine EphA1 by both structural and functional criteria. The chromosomal localization is shown to be syntenic to hEphA1 and the genomic organization also shows distinct features found in the hEphA1 gene. Functionally, in keeping with findings for the human homologue, both soluble recombinant and “native” mEphA1 show preferential binding to ephrin A1. However, we also observed significant binding to other A-type ligands as has been observed for other Eph receptors. We analysed the expression of mEphA1 mRNA by in situ hybridization on tissue sections. mEphA1 was expressed in epithelial elements of skin, adult thymus. kidney and adrenal cortex. Taken together with previous Northern blotting data these results suggest that mEphA1 is expressed widely in differentiated epithelial cells.

ELK4 neutralization sensitizes glioblastoma to apoptosis through downregulation of the anti-apoptotic protein Mcl-1

Glioma is the most common adult primary brain tumor. Its most malignant form, glioblastoma multiforme (GBM), is almost invariably fatal, due in part to the intrinsic resistance of GBM to radiation- and chemotherapy-induced apoptosis. We analyzed B-cell leukemia-2 (Bcl-2) anti-apoptotic proteins in GBM and found myeloid cell leukemia-1 (Mcl-1) to be the highest expressed in the majority of malignant gliomas. Mcl-1 was functionally important, as neutralization of Mcl-1 induced apoptosis and increased chemotherapy-induced apoptosis. To determine how Mcl-1 was regulated in glioma, we analyzed the promoter and identified a novel functional single nucleotide polymorphism in an uncharacterized E26 transformation-specific (ETS) binding site. We identified the ETS transcription factor ELK4 as a critical regulator of Mcl-1 in glioma, since ELK4 downregulation was shown to reduce Mcl-1 and increase sensitivity to apoptosis. Importantly the presence of the single nucleotide polymorphism, which ablated ELK4 binding in gliomas, was associated with lower Mcl-1 levels and a greater dependence on Bcl-xL. Furthermore, in vivo, ELK4 downregulation reduced tumor formation in glioblastoma xenograft models. The critical role of ELK4 in Mcl-1 expression and protection from apoptosis in glioma defines ELK4 as a novel potential therapeutic target for GBM.

Abstract 4247: TOLD MRI validation of reversal of tumor hypoxia in glioblastoma with a novel oxygen therapeutic

Background: Glioblastoma multiforme (GBM) is known to be a hypoxic tumor and hypoxia adversely affects response to radiation therapy. Dodecafluoropentane emulsion (DDFPe) on a weight basis, delivers over 100x as much oxygen as other higher molecular weight fluorocarbons and is under study as an oxygen therapeutic in GBM patients treated with chemo-irradiation. Tissue oxygen level dependent (TOLD) MRI is an oxygen sensitive imaging technique that is being used to study tumor re-oxygenation in these patients. Methods: With informed consent and IRB approval adult patients with GBM with residual tumor post-surgery were enrolled into the dose escalation phase of the study. Patients received DDFPe (2%w/vol DDFPe) at doses of 0.05 cc/kg, 0.1 cc/kg or 0.17 cc/kg I.V. 30-60 minutes prior to each fraction of radiation (2-Gy each, 30 fractions over 6 weeks). Temozolomide chemotherapy was given concurrently. Radiation was performed while the patients were breathing carbogen via a non-rebreathing circuit; the post-radiation MRI was performed while the patients were breathing oxygen. All patients underwent standard MR imaging at 1.5 Tesla. TOLD MRI was performed on patients at the higher two dose levels pre and post DDFPe on two separate occasions, days #1 and days #5 or 10 after initiation of radiation. Steady state free precession (SSFP) Look-Locker was used to obtain T1 maps. T1 maps were calculated from 3 axial slices obtained through tumor from either 3 or 4 repetitions. Results: TOLD MRI showed substantial decreases in T1 of tumor tissue after dosing with DDFPe and breathing oxygen (mean T1 decreased from 1501±454 ms to 1371±35 ms on day one and 1459±326 ms to 1183±271 ms on day 5 for the patient receiving 0.1 cc/kg DDFPe; from 1511±93 ms to 1250±85 ms on day 1 and from 1454±75 ms to 1281±77 ms on day 10 for the patient receiving 0.17cc/kg DDFPe), while inconsistent changes were found in contralateral normal brain (ranging from 863 to 1144 ms for both patients at all time points). The two different doses of DDFPe showed similar effect. Patients had second surgeries following completion of RT showing radiation necrosis consistent with enhanced effect of RT due to tumor re-oxygenation. Discussion and conclusion: The change in T1 (TOLD) with oxygen breathing after DDFPe administration indicated improved tumor oxygenation. The utility of TOLD MRI as a potential non-invasive prognostic imaging biomarker holds promise for precision medicine in terms of assessing tissue reoxygenation of hypoxic tumors. The ability to alter tumor oxygenation in GBM patients pre-treated with DDFPe could have important therapeutic implications. Dose expansion is currently underway with the dose of 0.1-cc/kg DDFPe. Figure: Representative T1 maps of two patients pre (air breathing) and post (oxygen breathing) DDFPe injection and radiation treatments. Arrows indicate tumors. Citation Format: Jason Lickliter, Jeremy Ruben, David B. Wilson, Heling Zhou, Ralph Mason, Evan C. Unger. TOLD MRI validation of reversal of tumor hypoxia in glioblastoma with a novel oxygen therapeutic. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4247.

An imatinib-only window followed by imatinib and chemotherapy for Philadelphia chromosome-positive acute leukemia: long-term results of the CMLALL1 trial

Abstract We report long-term results in 40 patients with Philadlephia chromosome-positive (Ph+) acute leukemia who received an imatinib monotherapy window to evaluate in vivo effects on BCR–ABL signaling prior to induction chemotherapy. The first 25 patients (cohort 1) received the LALA-94 protocol without further imatinib (newly diagnosed Ph+ acute lymphoblastic leukemia [ALL]) or induction chemotherapy followed by single-agent imatinib. Subsequent patients (cohort 2) continued imatinib concurrently with either LALA-94 (newly diagnosed Ph + ALL) or other intensive chemotherapy regimens. Cohort 2 had a complete response (CR) rate of 93% and 5-year survival of 69%. For newly diagnosed Ph+ ALL, survival was superior in cohort 2 compared with cohort 1. Toxicity was similar to that expected for chemotherapy alone. Among 10 evaluable patients, rapid loss of phospho-CRKL occurred during the imatinib window in seven patients (all achieved CR) and incomplete inhibition in three patients (none with CR). In summary, a pharmacodynamic window design permitted biomarker assessment of BCR–ABL targeting without compromising clinical outcomes.

Abstract CT305: Phase I studies of a selective cMet inhibitor AZD6094 (HMPL504/volitinib) in patients with advanced solid tumors

Background: Volitinib is a selective oral small molecule inhibitor of cMet kinase, which has demonstrated potent in vivo inhibitory effects on a variety of human tumor xenografts. Methods: Two phase I dose-escalation studies have been conducted in Australia (AU) and China (CN) in parallel to determine the maximum tolerated dose (MTD) or phase II Recommended Dose (P2RD), to evaluate pharmacokinetics (PK) profile, and to assess antitumor activity of Volitinib. Treatment was given orally in 21-day cycles until disease progression or unacceptable toxicity. Results: By July 2014 both studies completed dose-escalation phase. A total of 61 patients were enrolled (40 in AU and 21 in CN). Patients were treated with daily (QD) volitinib from 100mg to 1000mg or twice daily (BID) from 300 mg to 600mg. Median age at baseline was 63 years, and 60% patients were male in the AU study; whereas median age was 53 years, and 57% patients were male in the CN study. In both studies, the most common treatment related adverse events included nausea, vomiting, fatigue, peripheral edema and decreased appetite, mostly of grade (G) 1/2. Four patients experienced 5 dose limiting toxicities (DLTs) in the AU study: 1 G3 abnormal liver function test at 600mg QD, 1 G3 fatigue at 800mg QD, and 2 G3 fatigues and 1 G3 headache at 1000mg QD. One DLT of G3 fatigue at 600mg BID was reported in the CN study. The MTD for the QD regimen was identified as 800mg whereas the MTD for the BID regimen had not been reached in either study. 500mg BID was determined to be the P2RD as monotherapy based on the favorable benefit/risk profile demonstrated in both studies. In the AU study, 2 patients in the 600mg QD cohort and 1 patient in 1000mg QD cohort achieved partial response (PR). All 3 responders were papillary renal cell carcinoma patients. Two of the 3 responders remain PR with volitinib treatment of approximately 10 and 18 months respectively by July 2014. One CRC patient at 600mg QD achieved a 29% tumor reduction. Tumor sample analysis showed that all responders had both MET gene copy number increase (Chr7 gains or Met gene amplification) and high MET protein expression. Volitinib was rapidly absorbed with Tmax around 2∼4 hours and rapidly eliminated with half-life around 3∼7 hours in both studies. Both Cmax and AUC were roughly dose-proportional up to 800 mg QD and 500 mg BID. No obvious accumulation was found after 21-day of continuous QD or BID dosing. Drug exposure did not show racial difference between Caucasian and Asian patients. Conclusions: Volitinib was well tolerated up to 800 mg QD and 600 BID with acceptable safety profile. 500mg BID was determined to be the P2RD as monotherapy. Preliminary efficacy data demonstrated promising anti-tumor activity in patients with Met gene copy number increase or high protein expression. Volitinib demonstrated linear PK profile without marked drug accumulation. Further clinical studies are warranted. Citation Format: Ye Hua, Lin Shen, Hui Gan, Jason Lickliter, Michael Millward, Jianming Xu, Jian Wang, Yang Sai, Weiguo Su, Melanie M. Frigault, Chuan Qi. Phase I studies of a selective cMet inhibitor AZD6094 (HMPL504/volitinib) in patients with advanced solid tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr CT305. doi:10.1158/1538-7445.AM2015-CT305

Abstract 1197: EphA3 kinase ablation induces glioblastoma differentiation and prevents tumor progression

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Purpose: Eph receptors constitute the largest sub-family of receptor tyrosine kinases and interact with membrane-bound ligands termed ephrins. Eph and ephrins have many vital functions including cell adhesion, migration and axon guidance. Eph and ephrins have been found to be aberrantly expressed in many malignancies including brain tumor. The purpose of this study was to investigate EphA3 receptor function in the most common and aggressive form of brain tumor, Glioblastoma (GBM). Methods: Gene expression was investigated by Q-PCR, IHC and flow cytometry in high grade glioma (HGG) surgical specimens and primary derived serum free cell cultures. Targeted reduction of Eph expression was performed using both a constitutive and inducible shRNA system. Murine in-vivo studies were performed using both subcutaneous and orthotopic ‘intracranial’ xenografts in immuno-compromised animals. Signaling pathways were assessed by western blotting. Results: To establish whether the receptor tyrosine kinase EphA3 was over expressed in HGG we assessed 12 normal human brain specimens, 56 HGG specimens and 26 HGG primary cultures. EphA3 mRNA expression was negligible in normal brain while 30% of clinical specimens and 46% of primary cultured tumor cells expressed EphA3. EphA3 protein was also detected in HGG clinical specimens using IHC. To further investigate EphA3 function the receptor was down regulated using shRNA in an EphA3+ GBM neurosphere cell line. Constitutive and inducible down regulation of the EphA3 receptor resulted in initiation of neuronal and glial cell differentiation following activation of the ERK/MAPK pathway. A reduction in stem/progenitor cell proliferation was also observed following EphA3 knockdown by shRNA (46%) or by alternately inhibiting EphA3 function using soluble ephrin A5-Fc (33%). CFSE division tracking identified slower cell division in populations in which EphA3 signaling was attenuated. In-vivo studies were performed using a NOD/SCID mouse subcutaneous and intracranial xenograft model. Results highlighted a marked reduction in tumor formation in the EphA3 knockdown as opposed to control tumors. Subcutaneous control tumors formed with a median survival of 66 days while EphA3 knockdown animals survived beyond 100 days (p<0.05). Similar to the subcutaneous xenograft model a marked lack of intracranial tumor formation was observed when EphA3 was neutralized. Control mice formed large well vascularized invasive tumors with a median survival of 62 days. All EphA3 knockdown animals were free of tumor following autopsy at 145 days when the experiment was terminated (p<0.05). Importantly a mutant EphA3 rescue of the knockdown culture returned the tumorigenic potential of these cells. Conclusions: We propose EphA3, in part, regulates cancer stem cell self renewal and cell division rate in GBM and could prove a potential therapeutic target and marker of brain tumor initiating stem cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1197. doi:10.1158/1538-7445.AM2011-1197