Robyn T. Sussman

Convergence of Acquired Mutations and Alternative Splicing of CD19 Enables Resistance to CART-19 Immunotherapy

UNLABELLED The CD19 antigen, expressed on most B-cell acute lymphoblastic leukemias (B-ALL), can be targeted with chimeric antigen receptor-armed T cells (CART-19), but relapses with epitope loss occur in 10% to 20% of pediatric responders. We detected hemizygous deletions spanning the CD19 locus and de novo frameshift and missense mutations in exon 2 of CD19 in some relapse samples. However, we also discovered alternatively spliced CD19 mRNA species, including one lacking exon 2. Pull-down/siRNA experiments identified SRSF3 as a splicing factor involved in exon 2 retention, and its levels were lower in relapsed B-ALL. Using genome editing, we demonstrated that exon 2 skipping bypasses exon 2 mutations in B-ALL cells and allows expression of the N-terminally truncated CD19 variant, which fails to trigger killing by CART-19 but partly rescues defects associated with CD19 loss. Thus, this mechanism of resistance is based on a combination of deleterious mutations and ensuing selection for alternatively spliced RNA isoforms. SIGNIFICANCE CART-19 yield 70% response rates in patients with B-ALL, but also produce escape variants. We discovered that the underlying mechanism is the selection for preexisting alternatively spliced CD19 isoforms with the compromised CART-19 epitope. This mechanism suggests a possibility of targeting alternative CD19 ectodomains, which could improve survival of patients with B-cell neoplasms.

Chemotherapy-resistant side-population of colon cancer cells has a higher sensitivity to TRAIL than the Non-SP, a higher expression of c-Myc and TRAIL-receptor DR4

Cancer stem cells are resistant to chemotherapy and provide an important target for drug development. We found that, surprisingly, the dye-effluxing side population (SP) within SW480 human colon tumor cells, a population defined to possess stem cell characteristics, expresses a 10-fold higher level of pro-apoptotic TRAIL receptor DR4 as compared to non-SP cells. The TRAIL receptors are activated by the anti-tumor host immune system through the TRAIL ligand. SW480 SP-cells express similar levels of another TRAIL receptor (DR5), as non-SP cells. SP-cells from multiple tumorigenic human cell lines, which are most often resistant to chemotherapeutic agents such as etoposide, cisplatin and 5-FU, are more sensitive to TRAIL than non-SP cells. SP-cells express higher levels of c-Myc than non-SP cells which may explain their sensitivity to TRAIL. We have found c-Myc activates DR4 transcription through E-box DNA-response elements located in the DR4 promoter, thereby increasing the expression of cell-surface pro-apoptotic death receptors in TRAIL-resistant cell lines. TRAIL sensitivity of SP-cells may represent a safeguard against malignancy, and therefore, offers a therapeutic window and opportunity.

The epigenetic modifier Ubiquitin Specific Protease 22 (Usp22) regulates embryonic stem cell differentiation via transcriptional repression of Sex determining region Y-box 2 (Sox2)

Background: Ubiquitin-specific protease 22 (USP22) is a deubiquitylating enzyme with established biological functions in cancer cells. Results: USP22 drives differentiation of embryonic stem cells (ESCs) and represses sex-determining region Y-box 2 (SOX2) transcription. Conclusion: USP22 is induced during ESC differentiation to repress SOX2 transcription. Significance: Understanding the epigenetic programs that control changes in gene expression during the transition from self-renewal to differentiation. Pluripotent embryonic stem cells (ESCs) undergo self-renewal until stimulated to differentiate along specific lineage pathways. Many of the transcriptional networks that drive reprogramming of a self-renewing ESC to a differentiating cell have been identified. However, fundamental questions remain unanswered about the epigenetic programs that control these changes in gene expression. Here we report that the histone ubiquitin hydrolase ubiquitin-specific protease 22 (USP22) is a critical epigenetic modifier that controls this transition from self-renewal to differentiation. USP22 is induced as ESCs differentiate and is necessary for differentiation into all three germ layers. We further report that USP22 is a transcriptional repressor of the locus encoding the core pluripotency factor sex-determining region Y-box 2 (SOX2) in ESCs, and this repression is required for efficient differentiation. USP22 occupies the Sox2 promoter and hydrolyzes monoubiquitin from ubiquitylated histone H2B and blocks transcription of the Sox2 locus. Our study reveals an epigenetic mechanism that represses the core pluripotency transcriptional network in ESCs, allowing ESCs to transition from a state of self-renewal into lineage-specific differentiation programs.

USP22 regulates oncogenic signaling pathways to drive lethal cancer progression.

Increasing evidence links deregulation of the ubiquitin-specific proteases 22 (USP22) deubitiquitylase to cancer development and progression in a select group of tumor types, but its specificity and underlying mechanisms of action are not well defined. Here we show that USP22 is a critical promoter of lethal tumor phenotypes that acts by modulating nuclear receptor and oncogenic signaling. In multiple xenograft models of human cancer, modeling of tumor-associated USP22 deregulation demonstrated that USP22 controls androgen receptor accumulation and signaling, and that it enhances expression of critical target genes coregulated by androgen receptor and MYC. USP22 not only reprogrammed androgen receptor function, but was sufficient to induce the transition to therapeutic resistance. Notably, in vivo depletion experiments revealed that USP22 is critical to maintain phenotypes associated with end-stage disease. This was a significant finding given clinical evidence that USP22 is highly deregulated in tumors, which have achieved therapeutic resistance. Taken together, our findings define USP22 as a critical effector of tumor progression, which drives lethal phenotypes, rationalizing this enzyme as an appealing therapeutic target to treat advanced disease.

Identification of GPC2 as an Oncoprotein and Candidate Immunotherapeutic Target in High-Risk Neuroblastoma

We developed an RNA-sequencing-based pipeline to discover differentially expressed cell-surface molecules in neuroblastoma that meet criteria for optimal immunotherapeutic target safety and efficacy. Here, we show that GPC2 is a strong candidate immunotherapeutic target in this childhood cancer. We demonstrate high GPC2 expression in neuroblastoma due to MYCN transcriptional activation and/or somatic gain of the GPC2 locus. We confirm GPC2 to be highly expressed on most neuroblastomas, but not detectable at appreciable levels in normal childhood tissues. In addition, we demonstrate that GPC2 is required for neuroblastoma proliferation. Finally, we develop a GPC2-directed antibody-drug conjugate that is potently cytotoxic to GPC2-expressing neuroblastoma cells. Collectively, these findings validate GPC2 as a non-mutated neuroblastoma oncoprotein and candidate immunotherapeutic target.

Differential killing of CD56-expressing cells by drug-conjugated human antibodies targeting membrane-distal and membrane-proximal non-overlapping epitopes

ABSTRACT CD56 (NCAM, neural cell adhesion molecule) is over-expressed in many tumor types, including neuroblastoma, multiple myeloma, small cell lung cancer, ovarian cancer, acute myeloid leukemia, NK-T lymphoma, neuroendocrine cancer and pancreatic cancer. Using phage display, we identified 2 high-affinity anti-CD56 human monoclonal antibodies (mAbs), m900 and m906, which bound to spatially separated non-overlapping epitopes with similar affinity (equilibrium dissociation constant 2.9 and 4.5 nM, respectively). m900 bound to the membrane proximal fibronectin type III-like domains, whereas m906 bound to the N-terminal IgG-like domains. m906 induced significant down-regulation of CD56 in 4 neuroblastoma cell lines tested, while m900-induced downregulation of CD56 was much lower. Antibody-drug conjugates (ADCs) made by conjugation with a highly potent pyrrolobenzodiazepine dimer (PBD) exhibited killing activity that correlated with CD56 down-regulation, and to some extent with in vivo binding ability of the antibodies. The m906PBD ADC was much more potent than m900PBD, likely due to higher CD56-mediated downregulation and stronger binding to cells. Treatment with m906PBD ADC resulted in very potent cytotoxicity (IC50: 0.05–1.7 pM). These results suggest a novel approach for targeting CD56-expressing neuroblastoma cells. Further studies in animal models and in humans are needed to find whether these antibodies and their drug conjugates are promising candidate therapeutics.

Inhibition of the Single Downstream Target BAG1 Activates the Latent Apoptotic Potential of MYC

ABSTRACT Aberrant MYC expression is a common oncogenic event in human cancer. Paradoxically, MYC can either drive cell cycle progression or induce apoptosis. The latent ability of MYC to induce apoptosis has been termed “intrinsic tumor suppressor activity,” and reactivating this apoptotic function in tumors is widely considered a valuable therapeutic goal. As a transcription factor, MYC controls the expression of many downstream targets, and for the majority of these, it remains unclear whether or not they play direct roles in MYC function. To identify the subset of genes specifically required for biological activity, we conducted a screen for functionally important MYC targets and identified BAG1, which encodes a prosurvival chaperone protein. Expression of BAG1 is regulated by MYC in both a mouse model of breast cancer and transformed human cells. Remarkably, BAG1 induction is essential for protecting cells from MYC-induced apoptosis. Ultimately, the synthetic lethality we have identified between MYC overexpression and BAG1 inhibition establishes a new pathway that might be exploited to reactivate the latent apoptotic potential of MYC as a cancer therapy.

Enzymatic assays for assessing histone deubiquitylation activity

While the post-translational modification of histones by the addition of ubiquitin was discovered decades ago, it has only recently been appreciated that the dynamic regulation of histone ubiquitylation patterns is an important mechanism for controlling a variety of biological processes. The processes include transcription, the recognition and repair of genomic damage and DNA replication, among others. Enzymes that catalyze the addition of ubiquitin to histones, such as the polycomb family, have been well-studied. In contrast, the enzymes that remove ubiquitin from histones are less well understood. The assay strategies described here provide a platform for the thorough in vitro and in vivo analysis of histone deubiquitylation. In some cases, these poorly characterized enzymes are likely to provide new opportunities for therapeutic targeting and a detailed understanding of their biochemical and biological activities is a prerequisite to these clinical advances.

Abstract 685: GPC2 is an oncogene and immunotherapeutic target in high-risk neuroblastoma

Background: GD2-directed immunotherapeutic strategies have improved outcomes in neuroblastoma; however, the majority of patients treated suffer relapse and GD2 expression on pain fibers causes dose-limiting toxicities. Methods: To identify alternative cell surface immunotherapeutic targets, we compared high-risk neuroblastoma (n=126 tumors) and normal tissue RNA sequencing data (GTEx; n=7859 samples from 31 normal tissues) and prioritized genes by differential and absolute expression and cell surface prediction. Genes were further surveyed for somatic copy number gain and correlative expression with MYCN amplification. Differential protein expression and localization were confirmed in neuroblastoma primary tumors (n=98), patient-derived xenografts (n=32; PDXs), cell lines (n=23), and normal pediatric tissues (n=36). Cell lines were subjected to candidate gene gain and loss of function studies (n=11). Additional pediatric tumor RNA sequencing data was surveyed followed by confirmatory immunohistochemistry (IHC). Finally, candidate specific antibodies were isolated from a human Fab phage library and utilized for antibody-drug conjugate (ADC) engineering followed by cytotoxicity studies. Results: We identified 33 differentially expressed cell surface molecules from which we prioritized glypican-2 (GPC2) for validation given GPC2’s robust differential expression (log-fold change tumor vs. normal tissue = 1.71-9.22; p=1.99 x 10-9-1.88 x10-300), high-level absolute RNA expression (median FPKM=60), and frequent DNA copy number gain associated with higher GPC2 expression (35%, n=182 tumors; p Conclusions: GPC2 is an oncogene and immunotherapeutic target in neuroblastoma and potentially other cancers. Citation Format: Kristopher R. Bosse, Pichai Raman, Maria Lane, Robyn T. Sussman, Jo Lynne Harenza, Daniel Martinez, Sabine Heitzeneder, Zhongyu Zhu, Komal Rathi, Michael Randall, Laura Donovan, Sorana Morrissy, Doncho V. Zhelev, Yang Feng, Jennifer Hwang, Yanping Wang, Bruce Pawel, Tricia Bhatti, Mariarita Santi, Javed Khan, Michael Taylor, Dimiter S. Dimitrov, Crystal Mackall, John M. Maris. GPC2 is an oncogene and immunotherapeutic target in high-risk neuroblastoma [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 685. doi:10.1158/1538-7445.AM2017-685

Abstract PR02: GPC2 is a candidate immunotherapeutic target and putative oncogene in high-risk neuroblastoma and other pediatric cancers

Background: Children with high-risk neuroblastoma have a poor prognosis despite intensive multimodal chemoradiotherapy. While monoclonal antibodies targeting the disialoganglioside GD2 improve outcomes in neuroblastoma, this therapy is associated with significant “on target-off tumor” toxicities. Thus, a major challenge remains in identifying novel cell surface molecules that meet the stringent criteria for modern immunotherapeutics, including unique tumor expression compared to normal childhood tissues, and preferably that these cell surface molecules be required for tumor sustenance and thus may be less susceptible to immune escape mechanisms. Methods: Differentially expressed genes that represent putative cell surface immunotherapeutic targets were initially identified by comparing high-risk neuroblastoma RNA sequencing data (N=126 primary tumors) to paired normal tissue data (GTEx; N=25 unique normal tissues; N=1-313 replicate samples/tissue type). For further prioritization, gene sets were filtered by in silico cell surface prediction and by absolute RNA expression, and then by assessing primary tumor DNA copy number via SNP genotyping data (N=177). For prioritized genes, protein expression and cellular localization was confirmed by Western blot, immunohistiochemistry (IHC), immunofluroescence (IF) and membrane extraction techniques in neuroblastoma cell lines and primary tumors. For functional characterization, neuroblastoma cell lines (N=12) were subjected to both gain and loss of function studies for each candidate gene. Results: The initial transcriptome-based discovery effort identified 649 significantly differentially expressed genes (log-fold change tumor vs. normal >1 for each tissue; adjusted p p -10 ), high-level absolute RNA expression (median FPKM=57; 85% of tumors with FPKM >25) and consistent DNA copy number gain (31% of primary neuroblastomas; N=177) associated with significantly higher GPC2 expression ( p p Conclusions: GPC2 is a candidate cell surface immunotherapeutic target and putative oncogene in high-risk neuroblastoma. More globally, these data show that genome-wide transcriptome analysis integrated with genomic and functional validation can identify differentially expressed cell surface oncogenes that may be attractive immunotherapeutic targets. Development of a GPC2 directed chimeric antigen receptor is ongoing, and progress will be reported. This abstract is also presented as Poster A13. Citation Format: Kristopher R. Bosse, Pichai Raman, Robyn T. Sussman, Michael Randall, Dan Martinez, Zhongyu Zhu, Bruce Pawel, Tricia Bhatti, Javed Khan, Dimiter S. Dimitrov, Crystal Mackall, John M. Maris. GPC2 is a candidate immunotherapeutic target and putative oncogene in high-risk neuroblastoma and other pediatric cancers. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr PR02.