NF1 Deletions Research Articles

Abstract 5511: Characterization of molecular and functional consequences of somatic NF1 mutations in non-small cell lung cancers

Abstract Driver molecular alterations are found in >20% of non-small cell lung cancers (NSCLCs). They specifically target the RAS-MAPK pathway, including the EGFR, KRAS, and BRAF oncogenes. NF1 is a tumor suppressor gene that encodes neurofibromin, an inhibitor of the RAS-MAPK pathway. NF1 mutation detection is challenging owing to its large size, the presence of numerous pseudogenes, and the absence of mutation hotspot. According to The Cancer Genome Atlas data (TCGA), NF1 somatic mutations are found in ~15% of lung cancer. However, NF1 mutations in NSCLCs are not extensively explored in NSCLC to date. We hypothesized that NF1 alterations could define a specific NSCLC population with distinct clinical and molecular profiles. We performed NF1 analysis using next-generation sequencing in NSCLC surgical specimens with known KRAS, EGFR, TP53, BRAF, HER2, and PIK3CA status. We evaluated the specificities of NF1-mutated NSCLCs. Then, we established of NF1-mutated cellular models with different NF1 wild-type (WT) cell lines. We chose two NSCLC cell lines (A549 and NCI H-1703, ATCC), and one nontumorigenic human bronchial epithelial cell line (HBE4-E6/E7-C1, ATCC). Mono- and biallelic NF1 mutations were established using CRISPR-Cas9 and nickase CRISPR-Cas9 technologies. In vitro functional tests and drug screening were performed using these isogenic cell models. In our series of 138 lung adenocarcinoma specimens, 25 tumors showed NF1 mutations (18%) and 11 showed NF1 deletions (8%). NF1 mutations were rarely associated with other mutations. Most of patients with NF1 alterations were males (72%) and smokers (75%). Overall survival and disease-free survival were statistically better in patients with NF1 alteration patients (N=35) than in KRAS mutated patients (N=30) in univariate analysis. There were more NF1 mutations in patients treated by neoadjuvant chemotherapy (p = 0.01). Then, we established cellular models of NF1-mutated NSCLC, using nickase and CRISPR-Cas9 technology. In HBE4-E6/E7-C1 cells, mono- and biallelic NF1 mutations were generated. Loss of NF1 expression was confirmed by Western blot: partial and total loss-of-expression of neurofibromin was found in monoallelic and biallelic NF1 mutated cell lines, respectively. Using Western blot, we showed that pERK/ERK ratio was higher in NF1-mutated cell lines versus WT cell lines, confirming that NF1 loss-of-function triggered RAS-MAPK pathway activation. Transcriptome analysis confirmed this activation. Pharmacologic screen (including MEK inhibitors) in this isogenic NSCLC model will enable us to assess specific vulnerabilities due to NF1 mono- or biallelic mutations. Our results confirm that NF1 is frequently mutated and represents a distinct subtype of NSCLCs. A better comprehension of functional consequences of NF1 mutations, including mono- and biallelic alterations, may open new avenues for NSCLC therapy. Citation Format: Camille Tlemsani, Nicolas Pecuchet, Aurélia Gruber, Audrey Mansuet-Lupo, Diane Damotte, Marco Alifano, Jennifer Varin, Ingrid Laurendeau, Armelle Luscan, Benoit Rousseau, Beatrice Parfait, Ivan Bieche, Audrey Briand, Benoit Terris, Helene Blons, Karen Leroy, Dominique Vidaud, Michel Vidaud, Pierre Laurent-Puig, Eric Pasmant. Characterization of molecular and functional consequences of somatic NF1 mutations in non-small cell lung cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5511.

Primary intracranial spindle cell sarcoma with rhabdomyosarcoma-like features share a highly distinct methylation profile and DICER1 mutations.

Patients with DICER1 predisposition syndrome have an increased risk to develop pleuropulmonary blastoma, cystic nephroma, embryonal rhabdomyosarcoma, and several other rare tumor entities. In this study, we identified 22 primary intracranial sarcomas, including 18 in pediatric patients, with a distinct methylation signature detected by array-based DNA-methylation profiling. In addition, two uterine rhabdomyosarcomas sharing identical features were identified. Gene panel sequencing of the 22 intracranial sarcomas revealed the almost unifying feature of DICER1 hotspot mutations (21/22; 95%) and a high frequency of co-occurring TP53 mutations (12/22; 55%). In addition, 17/22 (77%) sarcomas exhibited alterations in the mitogen-activated protein kinase pathway, most frequently affecting the mutational hotspots of KRAS (8/22; 36%) and mutations or deletions of NF1 (7/22; 32%), followed by mutations of FGFR4 (2/22; 9%), NRAS (2/22; 9%), and amplification of EGFR (1/22; 5%). A germline DICER1 mutation was detected in two of five cases with constitutional DNA available. Notably, none of the patients showed evidenceof a cancer-related syndrome at the time of diagnosis. In contrast to the genetic findings, the morphological features of these tumors were less distinctive, although rhabdomyoblasts or rhabdomyoblast-like cells could retrospectively be detected in all cases. The identified combination of genetic events indicates a relationship between the intracranial tumors analyzed and DICER1 predisposition syndrome-associated sarcomas such as embryonal rhabdomyosarcoma or the recently described group of anaplastic sarcomas of the kidney. However, the intracranial tumors in our series were initially interpreted to represent various tumor types, but rhabdomyosarcoma was not among thetypical differential diagnoses considered. Given the rarity of intracranial sarcomas, this molecularly clearly defined group comprises a considerable fraction thereof. We therefore propose the designation "spindle cell sarcoma with rhabdomyosarcoma-like features, DICER1 mutant" for this intriguing group.

Developmental dosing with a MEK inhibitor (PD0325901) rescues myopathic features of the muscle-specific but not limb-specific Nf1 knockout mouse

Neurofibromatosis Type 1 (NF1) is a common autosomal dominant genetic disorder While NF1 is primarily associated with predisposition for tumor formation, muscle weakness has emerged as having a significant impact on quality of life. NF1 inactivation is linked with a canonical upregulation Ras-MEK-ERK signaling. This in this study we tested the capacity of the small molecule MEK inhibitor PD0325901 to influence the intramyocellular lipid accumulation associated with NF1 deficiency. Established murine models of tissue specific Nf1 deletion in skeletal muscle (Nf1MyoD−/−) and limb mesenchyme (Nf1Prx1−/−) were tested.Developmental PD0325901 dosing of dams pregnant with Nf1MyoD−/− progeny rescued the phenotype of day 3 pups including body weight and lipid accumulation by Oil Red O staining. In contrast, PD0325901 treatment of 4 week old Nf1Prx1−/− mice for 8 weeks had no impact on body weight, muscle wet weight, activity, or intramyocellular lipid. Examination of day 3 Nf1Prx1−/− pups showed differences between the two tissue-specific knockout strains, with lipid staining greatest in Nf1MyoD−/− mice, and fibrosis higher in Nf1Prx1−/− mice.These data show that a MEK/ERK dependent mechanism underlies NF1 muscle metabolism during development. However, crosstalk from Nf1-deficient non-muscle mesenchymal cells may impact upon muscle metabolism and fibrosis in neonatal and mature myofibers.

P53 and NF 1 loss plays distinct but complementary roles in glioma initiation and progression.

Malignant glioma is one of the deadliest types of cancer. Understanding how the cell of origin progressively evolves toward malignancy in greater detail could provide mechanistic insights and lead to novel concepts for tumor prevention and therapy. Previously we have identified oligodendrocyte precursor cell (OPC) as the cell of origin for glioma following the concurrent deletion of p53 and NF1 using a mouse genetic mosaic system that can reveal mutant cells prior to malignancy. In the current study, we set out to deconstruct the gliomagenic process in two aspects. First, we determined how the individual loss of p53 or NF1 contributes to aberrant behaviors of OPCs. Second, we determined how signaling aberrations in OPCs progressively change from pre-malignant to transformed stages. We found that while the deletion of NF1 leads to mutant OPC expansion through increased proliferation and decreased differentiation, the deletion of p53 impairs OPC senescence. Signaling analysis showed that, while PI3K and MEK pathways go through stepwise over-activation, mTOR signaling remains at the basal level in pre-transforming mutant OPCs but is abruptly up-regulated in tumor OPCs. Finally, inhibiting mTOR via pharmacological or genetic methods, led to a significant blockade of gliomagenesis but had little impact on pre-transforming mutant OPCs, suggesting that mTOR is necessary for final transformation but not early progression. In summary, our findings show that deconstructing the tumorigenic process reveals specific aberrations caused by individual gene mutations and altered signaling events at precise timing during tumor progression, which may shed light on tumor-prevention strategies.

Consideration of the haplotype diversity at nonallelic homologous recombination hotspots improves the precision of rearrangement breakpoint identification.

Precise characterization of nonallelic homologous recombination (NAHR) breakpoints is key to identifying those features that influence NAHR frequency. Until now, analysis of NAHR-mediated rearrangements has generally been performed by comparison of the breakpoint-spanning sequences with the human genome reference sequence. We show here that the haplotype diversity of NAHR hotspots may interfere with breakpoint-mapping. We studied the transmitting parents of individuals with germline type-1 NF1 deletions mediated by NAHR within the paralogous recombination site 1 (PRS1) or paralogous recombination site 2 (PRS2) hotspots. Several parental wild-type PRS1 and PRS2 haplotypes were identified that exhibited considerable sequence differences with respect to the reference sequence, which also affected the number of predicted PRDM9-binding sites. Sequence comparisons between the parental wild-type PRS1 or PRS2 haplotypes and the deletion breakpoint-spanning sequences from the patients (method #2) turned out to be an accurate means to assign NF1 deletion breakpoints and proved superior to crude reference sequence comparisons that neglect to consider haplotype diversity (method #1). The mean length of the deletion breakpoint regions assigned by method #2 was 269-bp in contrast to 502-bp by method #1. Our findings imply that paralog-specific haplotype diversity of NAHR hotspots (such as PRS2) and population-specific haplotype diversity must be taken into account in order to accurately ascertain NAHR-mediated rearrangement breakpoints.

Clinical Application of Whole Genome Array Improves the Diagnosis of Pediatric Brain Tumors.

Pediatric brain tumors are the leading cause of childhood cancer mortality. Recurring genetic abnormalities play an essential role in the diagnosis and prognosis of pediatric brain tumors. However, clinical workup has not routinely included whole genome assessment. Here, we present high resolution whole genome array results in 11 pediatric brain tumors. Array identified clinically relevant abnormalities in all samples. Copy number aberrations with targeted therapy implication, GOPC-ROS1 fusion, CDK4 amplification, and NF1 deletion, were detected in 3 cases. In addition, array detected recurring genetic abnormalities, including KIAA1549-BRAF fusion, 19q13.42 amplification, i(17q), and monosomy 6, which assisted accurate histological diagnosis in pediatric brain tumors. In conclusion, our results show that whole genome high-resolution array detects diagnostic and treatment-relevant copy number abnormalities in pediatric brain tumors.

Characterization of early communicative behavior in mouse models of neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1) is a common neurogenetic disorder caused by mutation of the NF1 gene, in which 80% of affected children exhibit cognitive and behavioral issues. Based on emerging evidence that NF1 may be an autism predisposition gene, we examined autism spectrum disorder (ASD)-relevant early communicative behavior in Nf1 mouse models and observed alterations in both models. The changes in early communicative behavior in Nf1 mutant mice should motivate further studies into the causative factors and potential treatments for ASD arising in the context of NF1.

Identification of an atypical microdeletion generating the RNF135-SUZ12 chimeric gene and causing a position effect in an NF1 patient with overgrowth.

Neurofibromatosis type I (NF1) microdeletion syndrome, which is present in 4-11% of NF1 patients, is associated with a severe phenotype as it is caused by the deletion of NF1 and other genes in the 17q11.2 region. The variable expressivity of the disease makes it challenging to establish genotype-phenotype correlations, which also affects prognosis and counselling. We here describe a 3-year-old NF1 patient with an atypical deletion and a complex phenotype. The patient showed overgrowth, café au lait spots, inguinal freckling, and neurological abnormalities. The extent of the deletion was determined by means of array comparative genomic hybridisation, and its breakpoints were isolated by means of long-range polymerase chain reaction. Sequence analysis of the deletion junction fragment revealed the occurrence of an Alu-mediated recombination that led to the generation of a chimeric gene consisting of three exons of RNF135 and eleven exons of SUZ12. Interestingly, the deletion shares a common RNF135-centred region with another deletion described in a non-NF1 patient with overgrowth. In comparison with the normal RNF135 allele, the chimeric transcript was 350-fold over-expressed in peripheral blood, and the ADAP2 gene located upstream of RNF135 was also up-regulated. In line with this, the deletion causes the loss of a chromatin TD boundary, which entails the aberrant adoption of distal cis-acting regulatory elements. These findings suggest that RNF135 haploinsufficiency is related to overgrowth in patients with NF1 microdeletion syndrome and, for the first time, strongly indicate a position effect that warrants further genotype-phenotype correlation studies to investigate the possible existence of previously unknown pathogenic mechanisms.

Tumours with class 3 BRAF mutants are sensitive to the inhibition of activated RAS.

Approximately 200 BRAF mutant alleles have been identified in human tumours. Activating BRAF mutants cause feedback inhibition of GTP-bound RAS, are RAS-independent and signal either as active monomers (class 1) or constitutively active dimers (class 2). Here we characterize a third class of BRAF mutants-those that have impaired kinase activity or are kinase-dead. These mutants are sensitive to ERK-mediated feedback and their activation of signalling is RAS-dependent. The mutants bind more tightly than wild-type BRAF to RAS-GTP, and their binding to and activation of wild-type CRAF is enhanced, leading to increased ERK signalling. The model suggests that dysregulation of signalling by these mutants in tumours requires coexistent mechanisms for maintaining RAS activation despite ERK-dependent feedback. Consistent with this hypothesis, melanomas with these class 3 BRAF mutations also harbour RAS mutations or NF1 deletions. By contrast, in lung and colorectal cancers with class 3 BRAF mutants, RAS is typically activated by receptor tyrosine kinase signalling. These tumours are sensitive to the inhibition of RAS activation by inhibitors of receptor tyrosine kinases. We have thus defined three distinct functional classes of BRAF mutants in human tumours. The mutants activate ERK signalling by different mechanisms that dictate their sensitivity to therapeutic inhibitors of the pathway.

Abstract 4671: Co-occurrence of alterations in the DNA damage repair genes synergize with uncontrolled proliferation and associate with very-poor prognosis in acute myeloid leukemia patients

Abstract Partner and localizer of BRCA2 (PALB2) plays a key role in the DNA damage repair (DDR) by recruiting BRCA1, BRCA2 and RAD51. Alterations in PALB2 were described in hereditary breast cancer and Fanconi Anemia (FA). Little is known in Acute Myeloid Leukemia (AML). Aim of the study is to define the frequency and interplay of PALB2 alterations with patterns of somatic mutations and copy number variants (CNVs) in AML. We genotyped 233 AML samples by Single Nucleotide Polymorphism array (SNP 6.0 and Cytoscan HD, Affymetrix) to detect CNVs. We also performed Whole Exome Sequencing (WES, Illumina) of 56 cases to detect single nucleotide variants and small indels (MuTect and Varscan 2.0). Differences in survival were assessed by Kaplan-Meier survival analysis and Breslow test. We detected PALB2 loss in 12/233 patients (5%), with a minimal common region of deletion of 6.6 Kb including exon 12, which encodes for the domain of interaction with RAD51, BRCA2 and POLH. PALB2 deletion were correlated with CN loss of MYH11, CREBBP, PLK1 and FANCA on chromosome (chr) 16 (p≤.005), deletions of TP53, NF1 and BRCA1 on chr 17 (p≤.004), 5q deletions (p≤.001) and loss of XPO1 on chr 2 (p≤.006). PALB2-loss patients were enriched for alterations in genes involved in the protein kinase pathway (p=5×10-4), JUN kinase activity (p=.0006) and, notably, in genes belonging to the chromosome breakage pathway (p=.001; TP53, BRCA1 and BRCA2). To identify mutations that co-operates with CN alterations, we integrated WES data of 56 AML patients. No mutations in PALB2 were detected. However, we identified mutations in other DDR genes, including FANCE, BRCA2, TP53 and BRCA1. Of note, alterations in the DDR genes frequently co-occurred with both mutations and CN loss of TP53, leading to homozygous loss of function of TP53. In addition, KRAS, IDH1, TET2 and BCOR mutations were mutually exclusive with PALB2 loss. Preliminary gene expression profiling data revealed a 2-fold upregulation of FANCA in samples with DDR genes alterations compared with patients with no alterations (p=.04), while the other genes of the pathway were not affected. Finally, patients harboring PALB2 loss had a poorer prognosis compared complex karyotype cases (p=.021). We here define a new subset of AML patients, characterized by the synergistic loss of key DDR genes, which may in turn result in FANCA over-expression as a mechanism to overcome the DDR deficit. Our data suggest that the leukemogenic process associated PALB2 loss integrates defects in the DDR together with enforced proliferative stimuli leading to uncontrolled proliferation of cells, which potentially accumulate genetic lesions. These results may help improve patient stratification and define ad hoc therapeutic strategies for this aggressive leukemia type. Supported by: ELN, AIL, AIRC, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. Citation Format: Antonella Padella, Giorgia Simonetti, Maria Chiara Fontana, Marco Manfrini, Giovanni Marconi, Anna Ferrari, Italo Faria do Valle, Marianna Garonzi, Cristina Papayannidis, Eugenia Franchini, Elisa Zuffa, Viviana Guadagnuolo, Samantha Bruno, Andrea Ghelli Luserna di Rorà, Emanuela Ottaviani, Daniel Remondini, Massimo Delledonne, Giovanni Martinelli. Co-occurrence of alterations in the DNA damage repair genes synergize with uncontrolled proliferation and associate with very-poor prognosis in acute myeloid leukemia patients [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 4671. doi:10.1158/1538-7445.AM2017-4671

Alternative outcomes of pathogenic complex somatic structural variations in the genomes of NF1 and NF2 patients.

Multiplex ligation-dependent probe amplification (MLPA) has been widely used to identify copy-number variations (CNVs), but MLPA's sensitivity and specificity in mosaic CNV detection are largely unknown. Here, we present two mosaic deletions identified by MLPA as NF1 deletion of exons 17-21 and NF2 deletion of exons 9-10. Through cDNA analysis, genomic breakpoint-spanning PCR and Sanger sequencing, we found however both NF1 and NF2 deletions are each composed of two consecutive deletions, which cannot be differentiated by MLPA. Importantly, these consecutive deletions are most likely originating from a single genomic rearrangement and have been preserved independently in different populations of cells.

Limitations of the Pax7-creERT2 transgene for driving deletion of Nf1 in adult mouse muscle.

Neurofibromatosis Type 1 (NF1) is an autosomal dominant genetic disorder that results in a variety of characteristic manifestations. Prior studies have shown reduced muscle size and global skeletal muscle weakness in children with NF1. This associated weakness can lead to significant challenges impacting on quality of life. Pre-clinical studies using a muscle-specific NF1 knockout mouse have linked this weakness to an underlying primary metabolic deficiency in the muscle. However, the neonatal lethality of this strain prevents analysis of the role of NF1 in adult muscle. In this study, we present the characterization of an inducible muscle-specific NF1 knockout strain (Nf1Pax7i f/f ) produced by cross breeding the Pax7-CreERT2 strain with the conditional Nf1flox/flox line. Tamoxifen dosing of 8-week old Nf1Pax7i f/f mice led to recombination of the floxed allele in muscle, as detected by PCR. Detailed phenotypic analysis of treated adult mice over 8 weeks revealed no changes in bodyweight or muscle weight, no histological signs of myopathy, and no functional evidence of distress or impairment. Subsequent analysis using the Ai9 Cre-dependent tdTomato reporter strain was used to analyse labelling in embryos and in adult mice. Cell tracking studies identified a lower than expected rate of integration of recombined satellite cells into adult muscle. In contrast, a high persistent contribution of embryonic cells that were Pax7+ were found in adult muscle. These findings indicate important caveats with the use of the Pax7-CreER T2 strain and highlight a need to develop new tools for investigating the function of NF1 in mature muscle.

A Genomic Predictive Signature for Rigosertib in Lower Risk MDS Derived By Integrating Clinical Response, Mechanism of Action Data and Simulation

Background: Lower risk non-del5q MDS patients (pts) have limited treatment options and majority pts are transfusion dependent. Erythropoiesis stimulating agents (ESA) are first line of therapy for LR-MDS pts but almost half are resistant to ESA or become resistant after responding. Hence, LR-MDS represents an area of unmet medical need for novel agents to improve hematopoiesis and reduce transfusion dependency. Rigosertib (RIG) blocks RAS-mediated activation of proteins containing a common RAS binding domain, or RBD (Athuluri-Divaker, et al; Cell 165; 643'16). Since many pathways include proteins that employ RBD (i.e. ras, raf PI3-Kinase), interference with RBD provides a novel approach to block proliferation. While RIG showed encouraging response in LR-MDS pts (Tycko, et al; Blood 2013; 122:2745), there is a need for a biomarker that can differentiate responders from non-responders. To correlate genomics with response to RIG, we employed computational tools to integrate molecular data and observed responses.Aim: To determine predictive value of a computational biology derived genomic signature in LR-MDS pts who are treated with RIG.Methods: Materials were derived from Phase I and II studies of RIG in LR-MDS pts. Efficacy was reported as transfusion independence. Bone marrow samples were collected and processed using standard methods. DNA was extracted from bone marrow mononuclear cells or T-cells (germline control) purified from peripheral blood samples. Exome sequencing was performed using Agilent SureSelect Human All Exon v4 - 51Mb kit and HiSeq 2000. Raw sequences were aligned to the human genome build hg19 using BWA software; SNPs and In/Dels analysis was performed using PICARD, SAMTOOLS and GATK.These results, entered into predictive computational biology software (Cellworks Group), generated disease-specific protein network maps using PubMed and other online resources. Digital drug simulations are conducted by measuring drug effect on a score: a composite of cell proliferation, viability and apoptosis. Each patient-specific network map was screened for RIG reduced progression in a dose-respondent manner. Computer predictions were blindly correlated with clinical outcomes.Results: Predicted response was blindly correlated with the clinical outcome for 19 pts: Results showed 16 matches and 3 mismatches; and the following predictive test statistics: PPV - 81.25%, NPV - 100%, Sensitivity - 100%: accuracy of the predictive test at 84.21%.Modeling of LR-MDS pts predicted amplification of genes MYC, FNTA on chromosome 8; amplification of genes LGALS3, AJUBA, MAX on chromosome 14; TIAM1 on chromosome 21; HMGCR, on chromosome 5; PDPK1, MAPK3, PLK1 on chromosome 16 and PIK3CA, RAF1 on chromosome 3 correlated with increased response to RIG. Aberrations that enhanced the downstream of RAS signaling via the RAF-ERK or PI3K-AKT-MTOR pathway increased the signal flow through the drug response paths. MYC amplification on chromosome 8 via increasing the Purine synthesis pathway and production of GTP increased the RAS-ERK and RHEB-MTOR signaling. MYC also enhanced CCND1 production/proliferation. Prenylation pathway genes FNTA and HMGCR amplification also affect RAS and RHEB; enhance the drug response pathways. Another pathway that was found to be significant in the drug response, reducing proliferation, was inhibition of PLK1 and AURKA. Amplification of AJUBA, PLK1 made this pathway more significant and improved drug response via inhibition of proliferation via TIAM1-RAC1-PAK1-AURKA-PLK1 and RAF-PLK1 pathway. Deletion of TP53 and NF1 on chromosome 17 did not correlate with response.MYC amplification was seen in LR and HR MDS pts and correlated with response to RIG. However, the computational biology method showed that MYC amplification alone is not a predictor for response since there were non-responder profiles with MYC amplification and responder profiles with MYC deletion that had other aberrations in genes on chromosomes 3, 14, 16 or 21.Conclusions: A predictive method that models multiple genomic abnormalities simultaneously showed greater than 80% correlation between RIG mediated protein network perturbations and clinical outcomes in LR-MDS. The method also explained lack of response and highlighted resistance pathways that could be targeted to recover sensitivity. We also established eligibility criteria for greater precision enrollment in future trials. [Display omitted] DisclosuresFruchtman:Onconova: Employment. Petrone:Onconova Therapeutics, Inc.: Employment. Zbyszewski:Onconova Therapeutics, Inc.: Employment. Hoffman:Onconova Therapeutics, Inc.: Employment. Vali:Cellworks Group: Employment. Singh:Cellworks Group: Employment. Usmani:Cellworks: Employment. Grover:Cellworks Group: Employment. Abbasi:Cellworks: Employment.

PI3K and MEK Inhibition in Hypodiploid Acute Lymphoblastic Leukemia

Introduction: Hypodiploid acute lymphoblastic leukemia (ALL) is a rare, high-risk subtype of B-ALL associated with poor outcome. Genomic analysis of over 130 Hypodiploid ALL patients defined the distinct genomic landscape of this disease, defining two principal subtypes including Ras-activating alterations and IKZF3 loss in near haploid tumors (24-31 chromosomes) and mutation of TP53 coupled with homozygous IKZF2 loss in low hypodiploid tumors (32-39 chromosomes). New therapies are needed to improve outcome of this disease, necessitating preclinical studies in hypodiploid tumors in vitro, ex vivo and in vivo.Hypothesis: The observation of frequent Ras pathway activating mutations in hypodiploid ALL tumors, as well as increased phosphorylation of downstream Ras signaling targets, suggests that these tumors may be susceptible to PI3K/MEK inhibition. Ras signaling target activation was observed in hypodiploid tumors irrespective of mutational status. Here we used cell lines and representative xenograft models to explore the therapeutic efficacy of inhibiting PI3K/MAPK signaling in hypodiploid ALL.Methods: The near haploid cell lines NALM-16 and MHH-CALL-2 and eight patient-derived xenograft (PDX) models (four near haploid and four low hypodiploid) were selected for preclinical studies. Xenografts were representative of the most common genomic lesions observed in hypodiploid ALL patients, including deletion of NF1, PAG1 and IKZF3 in near haploid and mutation of TP53 and IKZF2 deletion in low hypodiploid (Holmfeldt et al. Nat Genet 2013; Mullighan et al. Blood 2015). Next-gen sequencing of xenograft and diagnosis tumor DNA confirmed the presence of key genetic alterations in the xenografts. A lentiviral vector was used for stable expression of luciferase in xenograft tumors for in vivo disease burden monitoring. Cell lines were treated in vitro with PI3K, mTOR, MEK, CDK4/6 and BET inhibitors either as single agents, inhibitor combinations or combined with chemotherapeutic agents. Xenografts were treated ex vivo with single agents and in vivo with PI3K and MEK inhibitors with or without chemotherapy.Results: NALM-16 and MHH-CALL-2 cell lines were most sensitive to PI3K, mTOR and BET inhibitors with IC50 values in the sub-micromolar range (Fig. 1).Treatment of xenografts ex vivo showed near haploid and low hypodiploid tumors to be four-fold more sensitive to pan-PI3K inhibitors and PI3K/mTOR dual inhibitors compared to PI3K α and d isoform-specific inhibitors. Near haploid and low hypodiploid xenografts were sensitive to MEK and BET inhibition ex vivo, but MEK inhibitors showed weak activity in the near haploid cell lines. Treatment with CDK4/6 inhibitors either as single agents or in combination with MEK inhibitors revealed minimal efficacy. Changes in cell signaling upon treatment was assayed by phosphoflow (Fig. 2). Akt and Erk1/2 phosphorylation was variable between tumors, whereas 4E-BP1 and S6 phosphorylation was consistently elevated, suggesting strong signaling through mTOR pathways. Pan-PI3K and PI3K/mTOR dual inhibitors showed potent activity against hypodiploid ALL tumors in vitro and ex vivo, however PI3K inhibition alone was insufficient to eliminate tumors in vivo. Substantial weight loss in mice treated with PI3K/mTOR dual inhibitors limited prolonged study in vivo. MAPK inhibition showed activity in both near haploid and low hypodiploid tumors ex vivo and displayed a modest reduction in tumor growth as a single agent in vivo. Combining the MEK inhibitor GDC-0973 with dexamethasone treatment increased efficacy, but induced significantly more weight loss than either monotherapy.Conclusion: Treatment of hypodiploid cell lines and xenografts in vitro and ex vivo, respectively, with PI3K and MEK inhibitors showed promising results, however the anti-tumor effect in vivo appeared mostly cytostatic and inhibitors were not sufficient as single agents to kill the tumor cells. MEK inhibitors showed stronger activity than PI3K inhibitors on xenografts in vivo and ex vivo, but this was not significantly increased when combined with dexamethasone treatment. The notable resistance of hypodiploid cells to dexamethasone may underlie this limited efficacy in addition to the increase in GDC-0973 clearance observed upon combination. Our findings also highlight the need to consider the PK properties of agents in the preclinical setting, particularly in combinations. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Ral A, via activating the mitotic checkpoint, sensitizes cells lacking a functional Nf1 to apoptosis in the absence of protein kinase C.

Nf1 mutations or deletions are suggested to underlie the tumor predisposition of NF1 (neurofibromatosis type 1) and few treatments are available for treating NF1 patients with advanced malignant tumors. Aberrant activation of Ras in Nf1-deficient conditions is responsible for the promotion of tumorigenesis in NF1. PKC is proven to be an important factor in supporting the viability of Nf1-defected cells, but the molecular mechanisms are not fully understood. In this study, we demonstrate that the inhibition of protein kinase C (PKC) by 1-O-Hexadecyl-2-O-methyl-rac-glycerol (HMG, a PKC inhibitor) preferentially sensitizes Nf1-defected cells to apoptosis, via triggering a persistent mitotic arrest. In this process, Ral A is activated. Subsequently, Chk1 is phosphorylated and translocated to the nucleus. Silencing Ral A significantly blocks Chk1 nuclear translocation and releases HMG-treated Nf1-deficient cells from mitotic arrest, resulting in the reduction of the magnitude of apoptosis. Thus, our study reveals that PKC is able to maintain the homeostasis or viability of Nf1-defected cells and may serve as a potential target for developing new therapeutic strategies.

Digital PCR for discriminating mosaic deletions and for determining proportion of tumor cells in specimen.

Mosaicism, presence of a genetic feature in only a subpopulation of cells, is frequent in de novo genetic diseases. Among large deletions covering the NF1 tumor suppressor gene, the frequency of mosaicism can be as high as 40% in de novo patients. In this study, we demonstrate the high potential of digital PCR in detecting large NF1 deletions and in discriminating mosaic cases. By simultaneously assessing the NF1 gene and a reference gene RPP30, deletions could be unambiguously distinguished from non-deletion samples. Performing the same assay for mixed samples from a DNA with a deletion and a non-deletion DNA, a highly significant linear relation was obtained between the set-up ratio of the two samples and the measured ratio of NF1/RPP30 (P<0.0001), suggesting the high potential of digital PCR in discriminating mosaic deletions. Furthermore, digital PCR detects NF1 allele loss in a tumor specimen that was not detected by loss of heterozygosity analysis using polymorphic markers due to high content of non-tumor cells. Based on the measured ratio of NF1/RPP30, the proportion of the tumor cells in this specimen could be calculated as 25%. Our results demonstrate that dual-probe digital PCR is a simple and effective method for detecting deletions and for discriminating mosaic deletions. Furthermore, this method is sensitive for assigning somatic allele loss in tumor specimen and enables determining proportion of tumor cells.

Fine mapping of meiotic NAHR-associated crossovers causing large NF1 deletions.

Large deletions encompassing the NF1 gene and its flanking regions belong to the group of genomic disorders caused by copy number changes that are mediated by the local genomic architecture. Although nonallelic homologous recombination (NAHR) is known to be a major mutational mechanism underlying such genomic copy number changes, the sequence determinants of NAHR location and frequency are still poorly understood since few high-resolution mapping studies of NAHR hotspots have been performed to date. Here, we have characterized two NAHR hotspots, PRS1 and PRS2, separated by 20 kb and located within the low-copy repeats NF1-REPa and NF1-REPc, which flank the human NF1 gene region. High-resolution mapping of the crossover sites identified in 78 type 1 NF1 deletions mediated by NAHR indicated that PRS2 is a much stronger NAHR hotspot than PRS1 since 80% of these deletions exhibited crossovers within PRS2, whereas 20% had crossovers within PRS1. The identification of the most common strand exchange regions of these 78 deletions served to demarcate the cores of the PRS1 and PRS2 hotspots encompassing 1026 and 1976 bp, respectively. Several sequence features were identified that may influence hotspot intensity and direct the positional preference of NAHR to the hotspot cores. These features include regions of perfect sequence identity encompassing 700 bp at the hotspot core, the presence of PRDM9 binding sites perfectly matching the consensus motif for the most common PRDM9 variant, specific pre-existing patterns of histone modification and open chromatin conformations that are likely to facilitate PRDM9 binding.

Clinicopathologic implications of NF1 gene alterations in diffuse gliomas.

Recent studies have identified somatic alterations in the gene encoding for neurofibromin (NF1) in a subset of glioblastoma (GBM), usually associated with the mesenchymal molecular subtype. To understand the significance of NF1 genetic alterations in diffuse gliomas in general, we evaluated public databases and tested for NF1 copy number alterations in a cohort using fluorescence in situ hybridization. NF1 genetic loss (homozygous NF1 deletions or mutations with predicted functional consequences) was present in 30 (of 281) (11%) GBM and 21 (of 286) (7%) lower-grade gliomas in The Cancer Genome Atlas data. Furthermore, NF1 loss was associated with worse overall and disease-specific survival in the lower-grade glioma, but not GBM, Group in The Cancer Genome Atlas cohort. IDH1 or 2 mutations co-existed in lower-grade gliomas with NF1 loss (36%) but not in GBM. In our cohort studied by fluorescence in situ hybridization, NF1/17q (n = 2) or whole Ch17 (n = 3) losses were only identified in the GBM group (5/86 [6%]). Tumors with NF1/Ch17 loss were predominantly adult GBM (4/5); lacked EGFR amplification (0/4), strong p53 immunolabeling (1/5), or IDH1 (R132H) protein expression (0/5); but expressed the mesenchymal marker podoplanin in 4/5. NF1 genetic loss occurs in a subset of diffuse gliomas, and its significance deserves further exploration.

IM-11 * GBM-ASSOCIATED EXOSOME-PULSED AUTOLOGOUS DC VACCINE (DC-VEX(R)) INDUCES D-2 APOPTOTIC-STAGE AND BYSTANDER KILLING-EFFECT IN GBM OF THE MESENCHYMAL TYPE

INTRODUCTION: GBM is one of the most lethal cancers due to its resistance to apoptosis or type I PCD after chemo/radiotherapy. Due to its heterogenicity, we must treat this tumour with personalised cancer medicine. METHODS: We obtain relapsed GBM characterised by NF1 deletions and PTEN mutations of the mesenchymal type resistant to chemo/radiation Tx, and then we develop a GBM-model from which we isolate autologous dendritic cells and exosomes expressing CD9/CD91, both of which incubate ex-vivo in culture with granulocyte-macrophage colony stimulating factor,interleukin-4 and tumour necrosis factor-a. RESULTS: Post-treatment with the active specific immunotherapy via the GBM-associated antigen presenting dendritic cells, we observe humoral antibody responses and cellular immunity responses expanding circulating CD4 and CD8 CTLs which express TCRs for the specific GBM antigens which bind to the complex of class I MHC molecule and release IFN-γ,IL-2 and IL-6.RNA sequencing for genomic/epigenomic analysis has exhibited that exosomes carry coding and non-coding genes whose expression was different from the ones detected in the FFPE samples obtained from the initial biopsies exhibiting the most recent gene alterations which take place in the relapsed GBM. The active specific immunotherapy leads to D2 irreversible stage of apoptosis or type-I programmed cell death leading to a bystander killing effect after phagocytosis of the apoptotic bodies by the adjacent relapsed GBM causing their eradication. CONCLUSION: The DC-Vex® autologous dendritic cell vaccine due to pulsed exosomes, it can eradicate with humoral and cellular immunity pathways the relapsed chemo/radioresistant GBM of mesenchymal type.

Cooperative loss of RAS feedback regulation drives myeloid leukemogenesis.

RAS network activation is common in human cancers and, in acute myeloid leukemia (AML), achieved mainly through gain-of-function mutations in KRAS, NRAS, or the FLT3 receptor tyrosine kinase1. In mice, we show that premalignant myeloid cells harboring a KrasG12D allele retain low Ras signaling owing to a negative feedback involving Spry4 that prevents transformation. In humans, SPRY4 is located on chromosome 5q, a region affected by large heterozygous deletion that are associated with an aggressive disease in which gain-of-function RAS pathway mutations are rare. These 5q deletions often co-occur with chromosome 17 alterations involving deletion of NF1 - another RAS negative regulator - and TP53. Accordingly, combined suppression of Spry4, Nf1 and Trp53 produces high Ras signaling and drives AML in mice. Therefore, SPRY4 is a 5q tumor suppressor whose disruption contributes to a lethal AML subtype that appears to acquire RAS pathway activation through loss of negative regulators.