Zai Ahmad

The ALK(F1174L) mutation potentiates the oncogenic activity of MYCN in neuroblastoma.

The ALK(F1174L) mutation is associated with intrinsic and acquired resistance to crizotinib and cosegregates with MYCN in neuroblastoma. In this study, we generated a mouse model overexpressing ALK(F1174L) in the neural crest. Compared to ALK(F1174L) and MYCN alone, co-expression of these two oncogenes led to the development of neuroblastomas with earlier onset, higher penetrance, and enhanced lethality. ALK(F1174L)/MYCN tumors exhibited increased MYCN dosage due to ALK(F1174L)-induced activation of the PI3K/AKT/mTOR and MAPK pathways, coupled with suppression of MYCN pro-apoptotic effects. Combined treatment with the ATP-competitive mTOR inhibitor Torin2 overcame the resistance of ALK(F1174L)/MYCN tumors to crizotinib. Our findings demonstrate a pathogenic role for ALK(F1174L) in neuroblastomas overexpressing MYCN and suggest a strategy for improving targeted therapy for ALK-positive neuroblastoma.

Combined MYC and P53 defects emerge at medulloblastoma relapse and define rapidly progressive, therapeutically targetable disease

Summary We undertook a comprehensive clinical and biological investigation of serial medulloblastoma biopsies obtained at diagnosis and relapse. Combined MYC family amplifications and P53 pathway defects commonly emerged at relapse, and all patients in this group died of rapidly progressive disease postrelapse. To study this interaction, we investigated a transgenic model of MYCN-driven medulloblastoma and found spontaneous development of Trp53 inactivating mutations. Abrogation of p53 function in this model produced aggressive tumors that mimicked characteristics of relapsed human tumors with combined P53-MYC dysfunction. Restoration of p53 activity and genetic and therapeutic suppression of MYCN all reduced tumor growth and prolonged survival. Our findings identify P53-MYC interactions at medulloblastoma relapse as biomarkers of clinically aggressive disease that may be targeted therapeutically.

Molecular and In Vivo Characterization of Cancer-Propagating Cells Derived from MYCN-Dependent Medulloblastoma

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. While the pathways that are deregulated in MB remain to be fully characterized, amplification and/or overexpression of the MYCN gene, which is has a critical role in cerebellar development as a regulator of neural progenitor cell fate, has been identified in several MB subgroups. Phenotypically, aberrant expression of MYCN is associated with the large-cell/anaplastic MB variant, which accounts for 5-15% of cases and is associated with aggressive disease and poor clinical outcome. To better understand the role of MYCN in MB in vitro and in vivo and to aid the development of MYCN-targeted therapeutics we established tumor-derived neurosphere cell lines from the GTML (Glt1-tTA/TRE-MYCN-Luc) genetically engineered mouse model. A fraction of GTML neurospheres were found to be growth factor independent, expressed CD133 (a marker of neural stem cells), failed to differentiate upon MYCN withdrawal and were highly tumorigenic when orthotopically implanted into the cerebellum. Principal component analyzes using single cell RNA assay data suggested that the clinical candidate aurora-A kinase inhibitor MLN8237 converts GTML neurospheres to resemble non-MYCN expressors. Correlating with this, MLN8237 significantly extended the survival of mice bearing GTML MB allografts. In summary, our results demonstrate that MYCN plays a critical role in expansion and survival of aggressive MB-propagating cells, and establish GTML neurospheres as an important resource for the development of novel therapeutic strategies.

A study of PD-L1 expression in KRAS mutant non-small cell lung cancer cell lines exposed to relevant targeted treatments

We investigated PD-L1 changes in response to MEK and AKT inhibitors in KRAS mutant lung adenocarcinoma (adeno-NSCLC). PD-L1 expression was quantified using immunofluorescence and co-culture with a jurkat cell-line transfected with NFAT-luciferase was used to study if changes in PD-L1 expression in cancer cell lines were functionally relevant. Five KRAS mutant cell lines with high PD-L1 expression (H441, H2291, H23, H2030 and A549) were exposed to GI50 inhibitor concentrations of a MEK inhibitor (trametinib) and an AKT inhibitor (AZD5363) for 3 weeks. Only 3/5 (H23, H2030 and A549) and 2/5 cell lines (H441 and H23) showed functionally significant increases in PD-L1 expression when exposed to trametinib or AZD5363 respectively. PD-L1 overexpression is not consistent and is unlikely to be an early mechanism of resistance to KRAS mutant adeno-NSCLC treated with MEK or AKT inhibitors.

Abstract 2627: A study of dynamic changes in PD-L1 expression inKRASmutant adenocarcinoma of the lung exposed to signal transduction inhibitors

Aim MEK and AKT inhibitors inhibit signaling down steam of KRAS and are being evaluated as single agents or in combination with other anticancer drugs for the treatment of KRAS mutant adenocarcinoma of the lung (adeno-NSCLC). We investigated whether increased PD-L1 expression with functional T cell inhibitory consequences were a common mechanism of resistance to these drugs in this setting. Material and Methods A panel of 10 KRAS mutant adeno-NSCLC cell lines were studied. Cells were exposed to GI50 concentrations of a MEK (trametinib) and AKT (AZD5363) inhibitor for 6hrs, 24hrs and 3 weeks. PD-L1 expression on cell lines was studied by immunofluorescence. Immunofluorescence at various time points were expressed as a ratio of values in drug treated cells compared to untreated control. Functional consequences of PD-L1 expression were studied using a T cell cancer cell line (Jurkat) transfected with a luciferase reporter where co-culture of cancer cells expressing PD-L1 led to reduction in luminescence. Luminescence at various time points were expressed as ratio of luminescence values of drug treated cells compared to untreated controls. Results Following characterization of expression of PD-L1 in 10 KRAS mutant adeno-NSCLC cell lines, 5 cell lines with the highest expression of PD-L1 was chosen for functional assays (H441, H2291, H23, H2030 and A549). When exposed to trametinib for 3 weeks, 3/5 cell lines (H23, H2030 and A549) showed an statistically significant increase in expression of PD-L1 (range 1.1-2.4) however significant reduction of luciferase activity was only observed in H23 and H2030, 0.93 and 0.75, p= 0.018 and p=0.01 respectively. When exposed to AZD5363 for 3 weeks, 3/5 cell lines (H441, H23 and H2030) showed a statistically significant increase in PD-L1 expression (range 1.3-1.9) however significant reduction in luciferase reduction was seen in only H441 and H23, 0.86 and 0.76, p= 0.03 and p= 0.04 respectively. Conclusion Chronic exposure of KRAS mutant cell lines to MEK and AKT inhibitors cause minor increases in PD-L1 expression but this does not result in functional inhibition of T cells in all instances. Consequently, a functional increase in PD-L1 expression is unlikely to be a common mechanism of resistance to MEK and AKT inhibitors in KRAS mutant adeno-NSCLC. 1 Citation Format: Anna R. Minchom, Parames Thavasu, Zai Ahmad, Adam Stewart, Alexandros Georgiou, Mary ER O9Brien, Sanjay Popat, Jaishree Bhosle, Timothy A. Yap, Johann de Bono, Udai Banerji. A study of dynamic changes in PD-L1 expression in KRAS mutant adenocarcinoma of the lung exposed to signal transduction inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2627. doi:10.1158/1538-7445.AM2017-2627

Abstract 2243: Characterization of PD-L1 expression on circulating tumor cells (CTCs) isolated with a label-free inertial microfluidic system from advanced non-small cell lung cancer patients (NSCLC pts)

Background: NSCLC exhibits intratumor heterogeneity, with subpopulations of cells undergoing epithelial-mesenchymal transition. Such CTCs from NSCLC pts may be missed by the EpCAM-based CELLSEARCH® system (CS). The label-free ClearCell® FX inertial microfluidic system (FX) isolates CTCs based on size and inertia and may lead to more accurate CTC capture. PD-1/PD-L1 inhibitors have shown benefit in PD-L1+ NSCLC pts, but responses are still seen in PD-L1- pts, suggesting limitations in tumor PD-L1 testing. Also, PD-L1 testing on CTCs may be more practical than tumor rebiopsies and may provide insights into cancer heterogeneity. Methods: FX was validated with EpCAM-high and EpCAM-low cancer cell lines labeled with CellTracker dyes spiked into healthy volunteer (HV) blood for repeatability and reliability. Enriched cells were detected with the automated Bioview Duet imaging system for recovery (%). Identical spiking studies were conducted on CS for comparison. Antibodies for 5-color immunofluorescence (IF) (CK, CD45, DAPI, TTF1 [to detect lung adenocarcinoma cells], PD-L1) were optimized on EpCAM-high and EpCAM-low cell lines. 8ml of blood from NSCLC pts were enriched with FX for 5-color IF characterization. 7.5ml of blood from the same pts taken at identical timepoints were enumerated with CS for comparison. Blood was obtained from HV for CTC enumeration on FX and CS as controls. Results: Cell recovery of EpCAM-high cancer cell lines using FX produced similar counts to CS, e.g. lung NCI-H2066 cells: FX 64.9%±4.5 vs CS 74.4%±9.2 (p = 0.05); lung H1975 cells: FX 74.1%±11.9 vs CS 74.7%±10.7 (p = 0.91). In contrast, for EpCAM-low cells, a significant difference in cell recovery between FX and CS was seen, e.g. lung A549 cells: FX 59.3%±5.8 vs CS 26.1%±7.6 (p Conclusions: FX resulted in consistently high cell recovery rates regardless of EpCAM status. Higher CTC counts were isolated with FX vs CS in 90% of NSCLC pts. 40% of NSCLC adenocarcinoma pts had PD-L1+ TTF1+ CTCs, but PD-L1 CTC heterogeneity was seen in other pts, which may in part explain differences in responses to PD-1/PD-L1 inhibitors in NSCLC. Citation Format: Zai Ahmad, Jen Fraser-Fish, Rajiv Kumar, Bernadette Ebbs, Gemma Fowler, Penny Flohr, Mateus Crespo, Sanjay Popat, Jaishree Bhosle, Udai Banerji, Mary O’Brien, Johann S. de Bono, Timothy A. Yap. Characterization of PD-L1 expression on circulating tumor cells (CTCs) isolated with a label-free inertial microfluidic system from advanced non-small cell lung cancer patients (NSCLC pts). [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 2243.

Abstract 1488: In vivo magnetic resonance elastography in pediatric brain tumor models

Refined imaging strategies that could improve diagnosis and management of children with brain malignancies are urgently required. Pediatric brain malignancies possess distinct underlying biologies that discriminate them from adult tumors, even within a common neuropathological diagnosis such as glioblastoma (GBM). Magnetic resonance elastography (MRE) exploits the ability of MRI to visualize the propagation of shear waves resulting from vibrations applied to the cranium to map and quantify (kPa) brain tissue elasticity (Gd) and viscosity (Gl) in vivo. The altered viscoelastic properties of tumors, combined with the sensitivity of MRE for differences in tumor microstructure, establishes MRE as an attractive modality for the detection and differential diagnosis of brain malignancies. Here we aimed to determine whether the potential of MRE in the neuroradiological management of patients with brain malignancies could be extended to the pediatric population. The viscoelastic properties of two pediatric brain tumor models that faithfully emulate high risk childhood disease were investigated: i) Orthotopic D-212 MG pediatric supratentorial giant cell GBM (H3F3A wildtype, 11 year old patient) xenografts propagated in NCr nu/nu mice and ii) GTML/Trp53KI/KI transgenic mice that spontaneously develop aggressive, MYCN driven, p53 depleted, medulloblastomas. D-212 MG and GTML/Trp53KI/KI tumors were less elastic (lower Gd) and viscous (lower Gl), and therefore softer, than the surrounding brain tissue. Both tumor types were also significantly less elastic (D-212 MG Gd = 3.9±0.2; GTML/Trp53KI/KI Gd = 3.5±0.1) than the soft thalamic parenchyma in non-tumor-bearing mice (Gd = 5.9±0.2; p = 0.001 and p = 0.02, respectively, Mann-Whitney), in addition to being less viscous. Interestingly, GTML/Trp53KI/KI tumors demonstrated a bimodal distribution of Gd, which reflected the more marked transition between their relatively stiffer rim and the softer core compared with D-212 MG tumors. We demonstrate that two representative models of major high risk pediatric brain malignancies share the unique softness previously observed in adult brain tumor models, allowing their detection by MRE. This supports observations that pediatric GBMs are morphologically indistinguishable from adult GBMs. Although median Gd values were not sufficient to discriminate between the tumor types, the marked bimodal distribution of Gd in the GTML/Trp53KI/KI tumors was not apparent in D-212 MG GBM tumors. Whilst ongoing histopathological investigation into growth patterns, vascular, cellular and extracellular networks will aid elucidation of the pathological determinants of the bimodal signature, to date unique to GTML/Trp53KI/KI, these data reinforce the potential of MRE for the detection and differential diagnosis of pediatric brain malignancies based on their mechanical properties. Citation Format: Jin Li, Jessica K.R. Boult, Maria Vinci, Sergey Popov, Karen Barker, Zai Ahmad, Yann Jamin, Craig Cummings, Suzanne A. Eccles, Jeffrey C. Bamber, Ralph Sinkus, Louis Chesler, Chris Jones, Simon P. Robinson. In vivo magnetic resonance elastography in pediatric brain tumor models. [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 1488. doi:10.1158/1538-7445.AM2015-1488

MYC and TP53 defects interact at medulloblastoma relapse to define rapidly progressive disease and can be targeted therapeutically

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Disease recurrence following multi-modal therapy is the single most adverse event in medulloblastoma (MB). Currently >90% of relapsing patients die, accounting for ∼10% of childhood cancer deaths. MB is heterogeneous at diagnosis, comprising four molecular subgroups with distinct clinicopathological and molecular features and outcomes. The relevance of these features at relapse is unknown, making characterisation, modelling and targeted therapy of relapse biology essential to improve outcomes. However, relapsed MBs are not routinely biopsied in clinical practice. We undertook a first comprehensive investigation of the molecular, clinical and pathological features of 29 relapsed MBs and paired tumour samples taken at diagnosis, including the assessment of features with established significance at diagnosis (e.g. chromosome 17 and TP53 pathway status, MYC family ( MYC, MYCN ) gene amplification, polyploidy, C TNNB1 mutation and molecular subgroup status). Molecular subgroup was concordant at diagnosis and relapse, however evidence of alteration of all other features examined was found in relapsed tumours, with the majority of changes (30/44) representing acquired high-risk events. Most notably, MYC family gene amplifications and TP53 pathway defects commonly emerged in combination at relapse following conventional multimodal treatment ( P =0.02, 7/22, 32%) and predicted rapid progression to death ( P =0.016). These observations suggested aberrant activation of MYC family genes synergizes with TP53 inactivation in the genesis of biologically aggressive MB. To investigate any such relationship, we examined Trp53 status in our transgenic mouse model of spontaneously-arising MYCN-driven MB (GTML; Glt1-tTA/TRE-MYCN-Luc ). Somatic Trp53 mutations were found in 83% of tumors (n=10/12). Direct modelling of this interaction in GTML/ Trp53 KI/KI mice dramatically enhanced MB formation with 100% penetrance (43/43, median survival 47 days) in GTML/ Trp53 KI/KI versus 6% (3/50) in GTML; P <0.0001), faithfully mimicked clinicopathological characteristics of TP53-MYC family gene-associated relapsed human tumors, and validated the essential role of TP53 in potentiating the growth of MYCN-driven MB. Finally, therapeutic inhibition of Aurora-A kinase using MLN8237 in these tumours, and in derived neurospheres in vitro , promoted degradation of MYCN, reduced tumor growth and prolonged survival. In summary, while subgroup status remains stable, MBs display altered molecular, pathological and clinical features at relapse, and the emergence of combined TP53-MYC family gene defects is common following conventional therapy. Their association with rapid demise, coupled with their biological validation as driving and therapeutically exploitable events in a novel mouse MB model, strongly support further investigation and routine biopsy of relapse disease to drive future individualised therapeutic strategies. Citation Format: Rebecca M. Hill, Sanne Kuijper, Janet Lindsey, Ed C. Schwalbe, Karen Barker, Jessica Boult, Daniel Williamson, Zai Ahmad, Albert Hallsworth, Sarra Ryan, Evon Poon, Simon Robinson, Ruth Ruddle, Florence Raynaud, Louise Howell, Colin Kwok, Abhijit Joshi, Sarah Nicholson, Stephen Crosier, Stephen Wharton, Tom Jacques, Keith Robson, Antony Michalski, Darren Hargrave, Barry Pizer, Simon Bailey, Fredrik J. Swartling, Kevin Petrie, William A. Weiss, Louis Chesler, Steve Clifford. MYC and TP53 defects interact at medulloblastoma relapse to define rapidly progressive disease and can be targeted therapeutically. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-201. doi:10.1158/1538-7445.AM2014-LB-201

Abstract LB-87: A novel epigenetic blood test to monitor minimal residual disease in high-risk neuroblastoma.

Background: Cells present as minimal residual disease (MRD) following frontline therapy drive chemoresistant tumour relapse, thus their detection and targeting is a major priority in cancer therapy. Neuroblastoma (NB) is an aggressive neural crest-derived malignancy of infants and young children. A hallmark of NB is its clinical heterogeneity and the majority of infants with NB have a unique biology that results in spontaneous remission through differentiation in the absence of therapy, despite disease that may be disseminated to the liver, skin, and bone marrow at diagnosis. These infants (with stage 4S disease) have survival rates that exceed 95%. Children with high-risk NB, however, largely characterized by amplification of the MYCN oncogene, often have a disease that is therapy resistant. Although modest improvements in outcome have been achieved as a result of therapy intensification, 5-year event free survival in high-risk patients remains approximately 40-50%. Typically, high-risk neuroblastoma recurs after near complete remission is achieved and the development of effective strategies directed at MRD remains an unmet clinical need. Detection of MRD in neuroblastoma is usually performed using real-time quantitative (RQ)-PCR of neuroblastoma-specific transcripts. Tumor-selective mRNA markers levels are highly dependent on gene expression, which can vary between patients (by a factor of up 1000) and change during treatment. Furthermore, the applicability of a PCR target for MRD detection is also determined by its background expression in hematologic cells. Chromatin conformation patterns integrate the spatial arrangement of the chromatin and build a signature for the physiological status of the cell representing early changes in the genetic and epigenetic regulation. These signatures form the basis for a new technology, Episwitch ™, which can identify epigenetic patterns linked with cancer progression and aggressiveness. Results: Based on a screen of human NB cell lines containing amplification of MYCN or expression of high levels of MYCN protein versus NB cell lines lacking expression of MYCN, we have identified a panel of four NB-associated genetic markers: HDM2 , PHOX2B , TERT and TH . HDM2 and TERT are direct transcriptional targets of MYCN. This epigenetic signature is currently being validated in primary NB samples. An in vivo proof of concept study utilizing the equivalent mouse epigenetic signature is being performed in a relapse model of MYCN-driven NB. Conclusion: In this study we have developed and tested a novel blood test based on Episwitch ™ technology that discriminates MYCN-associated NB. Our data suggest this non-invasive test can be used to monitor remission/relapse status quantitatively with high sensitivity in high-risk MYCN-associated neuroblastoma, thus informing treatment decisions. Citation Format: Kevin Petrie, Megan Field, Zai Ahmad, Mehrnoush Dezfouli, Karen Barker, Magdalena Jeznach, Laura Glass, Albert Hallsworth, Yordan Sbirkov, Howard Womersley, Arthur Zelent, Philip Jordan, Alexandre Akoulitchev, Louis Chesler. A novel epigenetic blood test to monitor minimal residual disease in high-risk neuroblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-87. doi:10.1158/1538-7445.AM2013-LB-87

Abstract LB-387: MYCN dependence of tumor propagating cells in an orthotopic model of medulloblastoma

Medulloblastoma (MB) is the most common paediatric brain tumor and is a major cause of cancer death in children. MYCN expression is critical to cerebellar development, regulates the fate of neural progenitor cells (NPCs) and drives the expansion of tumor propagating cells (TPCs) thought to represent the cell-of-origin for this disease. To examine the role of MYCN in MB tumorigenesis, we established neurosphere cell lines enriched for putative TPCs from the cerebella of GTML mice (transgenic for doxycycline-regulable overexpression of MYCN and luciferase). These mice are predisposed to anaplastic/large-cell MB (AACR abstract LB-79). Neurospheres derived from GTML cerebella with positive luciferase imaging signals but no visible tumor tissue exhibited several characteristics consistent with enrichment for TPCs and/or NPCs. Spheroids proliferated efficiently in the absence of exogenous growth factor, exhibited self-renewal in clonal dilution assays, expressed high levels of Mycn protein and the NPC markers nestin and SOX2, and in response to serum, differentiated along neuronal and glial lineages (increased GFAP and III tubulin expression by immunofluorescence). Finally, orthotopic reimplantation at low titer ( Suppression of MYCN with doxycycline (confirmed by loss of luciferase imaging) caused growth arrest via a G1 blockade of neurospheres in vitro. Pretreatment of implanted spheres significantly reduced tumor penetrance ( Taken together, these data suggest that MYCN expression drives MB tumor formation by maintenance or expansion of a population of relatively undifferentiatedTPCs present in normal cerebella. This model represents a significant tool for further investigation of the role of MYCN in MB initiation and progression. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-387.