https://openworks.mdanderson.org/sumexp22/1146/thumbnail.jp

Abebe, Joshua A

Chattopadhyay, Chandrani

English

OpenWorks @ MD Anderson

IntroductionUveal Melanoma (UM) is a rare subtype ofmelanoma that grows from the melanocytes of theuveal tract of the eye. Although it’s more treatable ifthe primary tumor is isolated in the uvea, the poorprognosis of this disease is caused by itsmetastasis to the liver. The metastatic UM has onlyone FDA approved therapy and needs newtherapeutic strategies urgently.Previously we’ve identified that inhibition of IGF-1Rpathway using a targeting agent for insulin receptorsubstrate (IRS-1), inhibited UM cell survival andcell migration. IGF-1 is highly expressed in the liver(the primary site for UM metastasis), and thereceptor IGF-1R is overexpressed in UM. AlthoughNT157 (the IRS-1 inhibitor) showed inhibition ofUM cell survival, we were interested in developingcombination therapy strategies using this agent.Single agent therapies often do not work inmelanoma in the long run. In a high throughputcombination drug screen with NT157, we observedsynergistic effect with NT157 and the HDACinhibitor, Belinostat (Bel), in UM cell killing activity.To validate our findings from the drug screen, Iperformed combination treatments with NT157 andBelinostat on UM cell lines to determine theircombined effect on UM cell survival and also themechanism of such.Conclusion• IGF-1R pathway inhibition partially blockedUM cell survival• Unbiased high throughput drug screenindicated synergy between IGF-1R and HDACinhibition in UM cell killing activity.• This was validated in in vitro UM cells basedassays in the current study with nanomolardoses of each inhibitor• Dual targeting of IGF-1R and the HDACpathways improved UM cell killing over singleagent treatments.• Apoptosis was observed in low levels in thesingle agent-treated cells but even more forthe combination treatments.ReferencesFig. 3  Belinostat and NT157 combination shows high synergy scores from the combination drug screen.Apoptosis was induced in UM cells treated with NT 157 and Belinostat in combination Combined inhibition of IGF-1R pathway and HDAC blocks Uveal Melanoma cell survival and induces apoptosis.Joshua Abebe1, Fatima Khan2 and Chandrani Chattopadhyay2Undergraduate at the University of Notre Dame1, MDACC University Outreach Program1, Melanoma Medical Oncology, UT MD Anderson Cancer Center2Methods• We used multiple UM cell lines (MM28, MP38,92.1, MP41, MP65, MEL202, and MEL20-06-039)with representative genotypes, monosomy 3(metastatic UM genotype) and GNAQ/11 mutantsof UM in these cell lines.• The UM cell lines were cultured in RPMI 1640media supplemented with 10% FBS, Penicillin-streptomycin, glutamine and HEPES.• NT157, an inhibitor of IRS-1 and Belinostat, ahistone deacetylase inhibitor (HDAC) were bothpurchased from SelleckChem.• We assessed UM cell survival using MTT assaystreated on cells with various concentrations ofeach agent. We also did colony formation assaysfor a confirmatory clonogenic assay depicting UMcell survival.• Routine Western Blots (WB) were used formolecular analysis of the levels of protein markersspecific to each pathway and apoptosis.Fig. 4  Percent survival of monosomy 3 (MM28, MP41 and MEL20-06-039) and non-monosomy 3 (92.1, MEL202) UM cell lines under single agent and combination treatments ResultsHigh Throughput Drug Screen to predict novel combination therapy strategies for UMOur initial high throughput combination drug screenwith NT157 allowed us to predict a synergisticrelationship between IGF-1R and HDAC inhibition inUM cell killing. A TargetMol Approved Drug librarywas used to test 2054 approved drugs for UM cellkilling activity and eventually filtered down to 31candidates for a combination screen. Belinostat waschosen for further investigation as it showed a goodsynergy score with NT157 combination (Figure 3).UM cell survival is efficiently blocked withcombined treatment using Belinostat and NT157.Fig 1. Depiction of tumor formation in uveal tract of the eyeFig 2. UM tumor metastasis seen in the liver and image of the affected liver cellsMEL270OMM2.1OMM2.3NT157Combination treatment with NT157 and Belinostatsubstantially improved the inhibition of UM cellsurvival over individual single agent treatments ofUM cells as seen from MTT assays (Figure 4).We further confirmed the effect of the combinationtreatment on UM cell survival using colony formationassays (Figure 5).Fig. 5  UM cell lines with single agent and combination treatments in a  colony formation assay.The routine western blots indicated thatindividual protein markers of the respectivetarget pathways were reduced on treatmentswith IGF-1R pathway inhibitor (IRS-1) and HDACinhibitor (HDAC 3, Acetyl Histone H3) (Figure 6).Combination treatment also strongly inducedapoptosis in the UM cells as evidenced from theincrease in cleaved PARP levels (Figure 6).To validate these findings I tested the abovecombination treatment on UM cell survival. Iobserved that UM cells respond better to thecombination treatment as compared to thesingle agent treatments. I also tried to determinethe mechanism of improved UM cell killing fromthe combined treatment with apoptosis assays.Western blots for cleaved PARP indicatedapoptosis induction in UM cells as a result of thecombination treatments.The extent of response to the combinationtreatment is not the same in all cell lines andtherefore in the future we plan to investigate thereason behind such differential responses. Thiswould inevitably help improve the prognosis ofthis rare subtype of melanoma.• Economou MA, All-Ericsson C, Bykov V, et al.Receptors for the liver synthesized growth factorsIGF-1 and HGF/SF in uveal melanoma:intercorrelation and prognostic implications. ActaOphthalmol. 2008;86 Thesis 4: 20-25.• Reuveni H, Flashner-Abramson E, Steiner L, et al.Therapeutic destruction of insulin receptorsubstrates for cancer treatment. Cancer Res.2013;73: 4383-4394.• Histone Deacetylase Inhibitors Induce GrowthArrest and Differentiation in Uveal Melanoma.Solange Landreville, Olga A. Agapova, Katie A.Matatall, Zachary T. Kneass, Michael D. Onken,Ryan S. Lee, Anne M. Bowcock, and J. WilliamHarbour. Clin Cancer Res; 18(2) January 15, 2012.Fig. 6 Western blot analysis showing reduction of drug target levels /pathway marker proteins post treatment with single agents and in combination 020406080100120 MM28Mel20-06-03992.1MP41MEL202DiscussionUM being an extremely rare form of melanomaand very different from cutaneous melanomalacks established therapeutic measures. Ourpreliminary data shows IGF-1R inhibition to besuccessful in partial reduction of UM cellsurvival. Single agent therapies usually do notwork in melanoma in the long run, whichprompted our high throughput combination drugscreen with IGF-1R pathway inhibiting agentNT157. From the drug screen, we found thatinhibiting the HDAC pathway in combination withIGF-1R pathway can lead to synergy in UM cellkilling.

Combined inhibition of IGF-1R pathway and HDAC blocks Uveal Melanoma cell survival and induces apoptosis.

https://openworks.mdanderson.org/cgi/viewcontent.cgi?article=1146&context=sumexp22

Combined inhibition of IGF-1R pathway and HDAC blocks Uveal Melanoma cell survival and induces apoptosis.

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