Search

Maximum Productive frequency and clonality did not change significantly in blood or tumors post-anti-PD-L1 treatment.

The top differentially expressed genes from Figure 3

TCR repertoire of control PBS-treated tumors indicates rich and even clone distribution.

Only a subset of patients responds to immune checkpoint blockade (ICB) in melanoma. A preclinical model recapitulating the clinical activity of ICB would provide a valuable platform for mechanistic studies. We used melanoma tumors arising from an Hgftg;Cdk4R24C/R24C genetically engineered mouse (GEM) model to evaluate the efficacy of an anti–mouse PD-L1 antibody similar to the anti–human PD-L1 antibodies durvalumab and atezolizumab. Consistent with clinical observations for ICB in melanoma, anti–PD-L1 treatment elicited complete and durable response in a subset of melanoma-bearing mice. We also observed tumor growth delay or regression followed by recurrence. For early treatment assessment, we analyzed gene expression profiles, T-cell infiltration, and T-cell receptor (TCR) signatures in regressing tumors compared with tumors exhibiting no response to anti–PD-L1 treatment. We found that CD8+ T-cell tumor infiltration corresponded to response to treatment, and that anti–PD-L1 gene signature response indicated an increase in antigen processing and presentation, cytokine–cytokine receptor interaction, and natural killer cell–mediated cytotoxicity. TCR sequence data suggest that an anti–PD-L1–mediated melanoma regression response requires not only an expansion of the TCR repertoire that is unique to individual mice, but also tumor access to the appropriate TCRs. Thus, this melanoma model recapitulated the variable response to ICB observed in patients and exhibited biomarkers that differentiate between early response and resistance to treatment, providing a valuable platform for prediction of successful immunotherapy.Implications:Our melanoma model recapitulates the variable response to anti–PD-L1 observed in patients and exhibits biomarkers that characterize early antibody response, including expansion of the TCR repertoire.

Additional representative anti-PD-L1 treatment effect on tumor growth in melanoma bearing mice (Study 3).

Analysis of early tumor response to anti-PD-L1 treatment in Hgftg;Cdk4R24C/R24C melanoma bearing mice (Study 2).

PD-L1 and FOXP3 expression in treated tumors.

CD19 CAR T-cell therapy (CD19-CAR) has changed the treatment landscape and outcomes for patients with pre-B cell acute lymphoblastic leukemia (B-ALL). Unfortunately, primary non-response (PNR), sustained CD19+ disease and concurrent expansion of CD19-CAR, occurs in 20% of patients and is associated with adverse outcomes. Although some failures may be attributable to CD19 loss, mechanisms of CD19-independent, leukemia intrinsic resistance to CD19-CAR remain poorly understood. We hypothesized that PNR leukemias are distinct compared to primary sensitive (PS) leukemias and that these differences are present prior to treatment. We utilized a multi-omic approach to investigate this in 14 patients (7 PNR and 7 PS) enrolled in the PLAT-02 trial at Seattle Children's Hospital. Long-read PacBio sequencing identified one PNR where 47% of CD19 transcripts had exon 2 skipping, but other samples lacked CD19 transcript abnormalities. Epigenetic profiling discovered DNA hypermethylation at genes targeted by polycomb repressive complex 2 (PRC2) in embryonic stem cells. Similarly, ATAC-seq revealed reduced accessibility at these PRC2 target genes with a gain in accessibility of regions characteristic of hematopoietic stem cells and multi-lineage progenitors in PNR. Single cell RNA-seq and CyTOF analyses identified leukemic subpopulations expressing multi-lineage markers and decreased antigen presentation in PNR. We thus describe the association of a Stem Cell Epigenome (SCE) with primary resistance to CD19-CAR therapy. Future trials incorporating these biomarkers, with addition of multi-specific CAR T-cells targeting against leukemic stem cell or myeloid antigens, and/or combined epigenetic therapy to disrupt this distinct stem cell epigenome may improve outcomes of patients with B-ALL.

Fusion-positive alveolar rhabdomyosarcoma (FP-RMS) is an aggressive pediatric sarcoma driven primarily by the PAX3-FOXO1 fusion oncogene, for which therapies targeting PAX3-FOXO1 are lacking. We screened 62,643 compounds using an engineered cell line that monitors PAX3-FOXO1 transcriptional activity identifying a hitherto uncharacterized compound, PFI-63. RNA-seq, ATAC-seq, and docking analyses implicated histone lysine demethylases (KDMs) as its targets. Enzymatic assays confirmed the inhibition of multiple KDMs with highest selectivity for KDM3B. Structural similarity search of PFI-63 identified PFI-90 with improved solubility and potency. Biophysical binding of PFI-90 to KDM3B was demonstrated using NMR and SPR. PFI-90 suppressed the growth of FP-RMS in vitro and in vivo through downregulating PAX3-FOXO1 activity, and combined knockdown of KDM3B and KDM1A phenocopied PFI-90 effects. Thus, we report novel KDM inhibitors with highest specificity for KDM3B. Its potent suppression of PAX3-FOXO1 activity can be exploited as a new therapeutic approach for FP-RMS and other transcriptionally driven cancers.

SUMMARYDuring development, hematopoietic stem/progenitor cells (HSPCs) originate from a subset of hemogenic endothelial cells (ECs) through a process of endothelial-to-hematopoietic transition (EHT). This process is temporally restricted to short developmental windows and generates HSPC with distinct capabilities for hematopoiesis. Although it is generally thought that adult hematopoiesis is sustained by HSCs derived from hemogenic endothelium during development, some observations point to EHT persistence in the late fetus/perinatally. Here we use lineage tracking and bioinformatics analysis to assess the presence of hemogenic endothelial cells in the adult mouse. Our analysis identifies a subset of bone marrow-resident adult endothelial cells, characterized by the expression of VE-Cadherin and the transcription factor RUNX1, that produce CD45+ hematopoietic cells. This EHT generates hematopoietic progenitors, and mature myeloid and lymphoid cells in the adult mouse. Our results reveal the identification of a distinct source of adult blood.

Human papilloma virus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) patients have a poor prognosis and most patients with metastatic disease are resistant to pembrolizumab. We recently showed that depletion of SET and MYND domain protein 3 (SMYD3), a protein methyltransferase, induces upregulation of multiple type I IFN response genes in HPV-negative HNSCC human and mouse cells in vitro. Systemic treatment of flank HPV-negative mouse oral carcinoma 1 (MOC1) tumors with Smyd3 anti-sense oligonucleotides (ASOs) induced influx of CD8+ T-cells, and combined Smyd3 ASOs and anti-PD-1 treatment induced tumor growth restraint, with 75% of tumors cured or reduced and 25% of tumors escaping. Here, we aim to investigate the changes in the tumor microenvironment (TME) of MOC1 tumors treated with Smyd3 ASOs and in combination with anti-PD-1, and to elucidate mechanisms of resistance. MOC1 tumors treated with control or Smyd3 ASOs (n=3 per group) were harvested and single-cell RNA-seq was conducted. GeoMx, a high-plex spatial proteomic assay, was utilized on FFPE sections of MOC1 tumors treated with control or Smyd3 ASOs with or without anti-PD-1 (n=2 per group). To decipher mechanisms of resistance, single-cell RNA-seq of MOC1 tumors that responded or not to combination Smyd3 ASOs and anti-PD-1 was conducted. 26926 cells from six samples were available for single-cell RNA-seq analysis of control versus Smyd3 ASO treated tumors. UMAP analysis identified distinct immune-, stroma- and cancer-cell populations. Comparative analysis of the single-cell Smyd3 mRNA expression revealed downregulation of Smyd3 mRNA in CD8+ and CD4+ T-cells, B-cells, NK-cells and cancer cells, but not in myeloid cells, endothelial cells and fibroblasts, implying differential uptake of Smyd3 ASOs in the TME. CD8+ T-cells in the Smyd3 ASO treated tumors exhibited higher expression levels of Gzma and Gzmb, indicating enhanced cytotoxic activity. Colony stimulating factor 1 (Csf1), which is important for the differentiation of macrophages, was upregulated in CD8+ T-cells of Smyd3 ASO treated tumors. Single-cell RNA-seq of a non-responder versus a responder MOC1 tumor yielded 9170 and 11410 cells respectively, with more than a two-fold increase in the ratio of neutrophils and a two-fold decrease for CD8+ T-cells comparing the non-responder to responder tumor. Accordingly, cancer cells, CD8+ T-cells and macrophages of the non-responder tumor exhibited higher expression of Cxcl2, a chemokine that attracts PMNs. Differential gene expression analysis in all cell subsets in control versus Smyd3 ASO and responder versus non-responder tumors is ongoing. This work will elucidate the TME changes induced by Smyd3 ASOs in MOC1 tumors, and will reveal potential mechanisms of resistance to the Smyd3 ASOs and anti-PD-1 combination, enabling translation of this therapeutic approach in HPV-negative HNSCC. Citation Format: Malhar Patel, Daniel Tsai, Abdalla Abdelmaksoud, Noemi Kedei, Maria Hernandez, Baktiar Karim, Vassiliki Saloura. Dissecting the effects of Smyd3 depletion in the tumor microenvironment of HPV-negative head and neck squamous cell carcinoma mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2320.

Background: Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. Despite the survival rate of 90% for newly diagnosed children with ALL, the outcome for relapsed patients is historically poor with a less than 30% survival. CD19 CAR T-cell therapy (CART19) has shown remarkable response rates, between 80-90% in relapsed/refractory disease. Little is known about antigen-independent factors that predict initial resistance to CART19. We hypothesized that leukemias that are resistant to CART19 are distinct from sensitive leukemias and that these differences can be detected prior to therapy. Methods: To interrogate differences between resistant and sensitive leukemias, we obtained pre-treatment bone marrow aspirates (BMAs) from patients enrolled in a clinical trial at Seattle Children’s Hospital (PLAT-02). Samples were categorized based on patient response, with non-response defined as not achieving and maintaining minimal residual disease negativity at Day +63. Our study included 7 resistant and 7 sensitive leukemias as controls. We performed whole exome sequencing, bulk RNA-seq, PacBio-seq of the CD19 locus, array-based methylation, ATAC-seq, scRNA-seq, and CyTOF. Results: We found that non-response to CART19 is independent of leukemic subtype. Despite blasts being CD19+ in all patients by flow cytometry, we identified alternative splicing of CD19 in one non-responder, while the remaining non-responders expressed high levels of wildtype CD19. We discovered a distinctive DNA methylation pattern in the non-responders characterized by hypermethylation of PRC2 targets in embryonic and cancer stem cells (p = 8.15E-25) Furthermore, using gene set enrichment analysis of ATAC-seq data, we found increased accessibility of chromatin at regions associated with stem cell proliferation (NES = 2.31; p < 0.0001) and cell cycling (NES = 2.27; p < 0.0001). We found a greater similarity between accessibility patterns of non-responders to hematopoietic progenitors, including hematopoietic stem cells (p = 0.037) and common myeloid progenitors (p = 0.047). These findings were supported by an increased frequency of cell subpopulations expressing a multi-lineage phenotype (CD19, CD20, CD33, CD34; p = 0.009). Moreover, we find decreased expression of antigen presentation and processing pathways across all leukemic cells relative to responders (p = 0.0001). Conclusions: This study, one of the most comprehensive multi-omic analyses of samples from patients treated with CAR T-cells, identified resistance mechanisms that can be detected prior to treatment. We report the novel association of a stem cell phenotype, lineage plasticity, and decreased antigen presentation with resistance. These results support further refinement of eligibility for CART19 for children with leukemia and highlights the need for alternative of complimentary approaches for these patients. Citation Format: Katherine E. Masih, Rebecca Gardner, Hsien-Chao Chou, Abdalla Abdelmaksoud, Young K. Song, Luca Mariani, Vineela Gangalapudi, Berkley E. Gryder, Ashley Wilson, Serifat O. Adebola, Benjamin Z. Stanton, Chaoyu Wang, Xinyu Wen, Gregoire Altan-Bonnet, Michael C. Kelly, Jun S. Wei, Martha L. Bulyk, Michael C. Jensen, Rimas J. Orentas, Javed Khan. Multi-omic analysis identifies mechanisms of resistance to CD19 CAR T-cell therapy in children with acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3581.

Glioma is the most common primary malignant brain tumor with a poor prognosis. Immune checkpoint inhibitors have been of great interest in investigation of glioma treatments. Here, we report single-cell transcriptomic analyses of two tumor areas from an oligodendroglioma taken from a patient who had multiple tumor recurrences, following several chemotherapies and radiation treatments. The patient subsequently received nivolumab and was considered have disease progression based on conventional diagnostic imaging after two cycles of treatment. He underwent a debulking surgical resection and pathological diagnosis was recurrent disease. During the surgery, tumor tissues were also collected from the enhancing and non-enhancing areas for a scRNAseq analysis to investigate the tumor microenvironment of these radiographically divergent areas. The scRNAseq analysis reveals a plethora of immune cells, suggesting that the increased mass observed on MRI may be partially a result of immune cell infiltration. The patient continued to receive immunotherapy after a short course of palliative radiation and remained free of disease progression for at least 12 months after the last surgery, suggesting a sustained response to immunotherapy. The scRNAseq analysis indicated that the radiological progression was in large part due to immune cell infiltrate and continued immunotherapy led to a positive clinical outcome in a patient who would have otherwise been admitted to hospice care with halting of immunotherapy. Our study demonstrates the potential of scRNAseq analyses in understanding the tumor microenvironment, which may assist the clinical decision-making process for challenging glioma cases following immunotherapy.

Only a subset of patients responds to immune checkpoint blockade (ICB) in melanoma. A preclinical model recapitulating the clinical activity of ICB would provide a valuable platform for mechanistic studies. We used melanoma tumors arising from an Hgftg;Cdk4R24C/R24C genetically engineered mouse (GEM) model to evaluate the efficacy of an anti–mouse PD-L1 antibody similar to the anti–human PD-L1 antibodies durvalumab and atezolizumab. Consistent with clinical observations for ICB in melanoma, anti–PD-L1 treatment elicited complete and durable response in a subset of melanoma-bearing mice. We also observed tumor growth delay or regression followed by recurrence. For early treatment assessment, we analyzed gene expression profiles, T-cell infiltration, and T-cell receptor (TCR) signatures in regressing tumors compared with tumors exhibiting no response to anti–PD-L1 treatment. We found that CD8+ T-cell tumor infiltration corresponded to response to treatment, and that anti–PD-L1 gene signature response indicated an increase in antigen processing and presentation, cytokine–cytokine receptor interaction, and natural killer cell–mediated cytotoxicity. TCR sequence data suggest that an anti–PD-L1–mediated melanoma regression response requires not only an expansion of the TCR repertoire that is unique to individual mice, but also tumor access to the appropriate TCRs. Thus, this melanoma model recapitulated the variable response to ICB observed in patients and exhibited biomarkers that differentiate between early response and resistance to treatment, providing a valuable platform for prediction of successful immunotherapy. Implications: Our melanoma model recapitulates the variable response to anti–PD-L1 observed in patients and exhibits biomarkers that characterize early antibody response, including expansion of the TCR repertoire.

Background: Acute lymphoblastic leukemia (ALL) is the most common childhood cancer with a peak incidence between 3-5 years of age. Despite the improved survival rate of 90% for newly diagnosed children with ALL, the outcome for patients who relapse is poor with a less than 30% survival. CD19 CAR T-cells have shown remarkable response rates between 80-90% in relapsed/refractory disease. Little is known about antigen-independent factors that predict initial resistance to CD19 CAR T-cell therapy. We hypothesized that leukemias that are resistant to CD19 CAR T-cell therapy are distinct from sensitive leukemias and that these differences can be detected prior to therapy. Methods: To interrogate differences in the genomes, transcriptomes, intratumoral heterogeneity, and epigenetic landscapes between resistant and sensitive leukemias, we obtained pre-treatment bone marrow aspirates (BMAs) from patients enrolled in a clinical trial at Seattle Children's Hospital (PLAT-02). Samples were categorized based on patient response and included 7 resistant (2 with matched post-treatment) and 7 sensitive leukemias as matched controls. We performed whole exome sequencing, bulk RNA-seq, array-based methylation, scRNA-seq, CyTOF, ATAC-seq, and H3K27ac ChIP-seq. Additionally, we established CD19 CAR T resistant and sensitive patient derived xenografts (PDXs) of these leukemias, which are currently being used for in vivo modelling of these diseases. Results: We identified mutations and fusions in known epigenetic modifiers, including CREBBP and EP300, in all resistant leukemias. ATAC-seq revealed relatively more accessible chromatin in resistant patients in comparison to sensitive (16,082 vs. 6,070 peaks). The combination of altered epigenetic modifiers and increased chromatin accessibility is suggestive of epigenetic plasticity. Motif analysis of these differentially accessible peaks showed an enrichment for AP-1 binding proteins, particularly JUN and its partners, which provides a mechanism to avoid caspase-induced apoptosis, the downstream effect of cytotoxic T-cell killing. Methylation array and expression data indicate promoter hypermethylation and lowered expression of JUN pathway genes in sensitive leukemias. CyTOF identified a subpopulation with characteristics of both myeloid and lymphoid lineages in one sample that may represent a resistant population of cells selected for under CAR T-cell pressure. Analysis of the marrow microenvironment indicates that patients with sensitive leukemias have increased markers of T-cell activation, suggestive of more robust T-cells capable of rejecting the leukemias. Conclusions: This study establishes one of the most integrative and comprehensive profiling approaches for patient samples. We have shown the association of epigenetic plasticity, JUN pathway upregulation, and decreased T-cell activation with resistance to CD19 CAR T-cell therapy. We are currently validating these results by modelling our cohort's therapy resistance in vivo. Citation Format: Katherine E. Masih, Abdalla Abdelmaksoud, David Milewski, Berkley E. Gryder, Rebecca Gardner, Ashley L. Wilson, Benjamin Z. Stanton, Young K. Song, Chaoyu Wang, Xinyu Wen, Serifat Adebola, Zachary Rae, Adam Cheuk, Justin Lack, Gregoire Altan-Bonnet, Michael Kelly, Jun Wei, Michael Jensen, Rimas J. Orentas. Increased epigenetic plasticity and tumor heterogeneity predict resistance to CD19 CAR T cell therapy in pre-treatment pediatric acute lymphoblastic leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-141.

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer with a peak incidence at 3-5 years of age. Despite the improved survival rate of 90% for newly diagnosed children with ALL, the outcome for patients with relapsed disease is poor with a less than 30% overall survival. CD19 CAR T-cell therapy has shown remarkable response rates in relapsed/refractory disease. Long-term survival analysis has shown that initial response rates exceed 80%. However, durable response rates at one year are closer to 40%. Little is known about factors predicting durable response to CAR T therapy. We hypothesize that patients with CD19 CAR T-cell resistant ALL have a distinct disease compared to responders to therapy that can be identified in pretreatment leukemia. Utilizing advanced genomic, epigenetic, proteomic, and single-cell (sc) techniques, we characterized patient bone marrow aspirates (BMA) to identify mechanisms of resistance. Patients enrolled in PLAT-02 at Seattle Children’s Hospital were categorized according to the durability of their response to CD19 CAR T therapy. To characterize the molecular and genomic alterations specific to the therapy-resistant ALLs, we performed comprehensive analyses on pre-treatment therapy-resistant and sensitive BMAs using whole-exome sequencing, RNA- BMAs seq, scRNA-seq, sc B cell receptor (BCR)-seq, methylation array, H3K27ac ChIP-seq, ATAC-seq, and CyTOF. Additionally, we developed murine patient-derived xenografts (PDXs) for future studies. Initial mutation analyses revealed 5 hotspot mutations (ABL1, 2 x KRAS, IKZF1, and EP300) and actionable fusion (2 ABL1, 2 ETV6, 2 ETV5, KMT2A). Interestingly, we identified a KMT2A-AFF1 fusion in a sensitive leukemia, which has been demonstrated to predispose patients to CD19 CAR T resistance through lineage switching. Additionally, we identified a novel CREBBP-fusion in leukemias resistant to CD19 CAR T-induced B-cell aplasia. Alterations of CREBBP have previously been associated with ALL that is refractory to conventional therapies. Integrated gene expression and epigenetic analyses are ongoing to identify genes or pathways associated with resistant disease. scRNA- and scBCR-seq data are being analyzed and integrated with CyTOF analyses to detect mixed lineage and gene expression-based heterogeneity that may predict clonal selection by CAR T pressure. Finally, we developed and genetically analyzed murine PDXs for 64% of the patient samples, establishing a valuable resource for future studies and developing novel therapies for resistant leukemias. This study is one of the most integrative and comprehensive genomic profiling approaches to identify the molecular traits of therapy-resistant ALL in patient samples. We hope to identify and develop crucial biomarkers predicting responsiveness to CAR T-cell therapy. Citation Format: Katherine E. Masih, Rebecca Gardner, Berkley E. Gryder, Abdalla Abdelmaksoud, Ashley Wilson, Serifat Adebola, Benjamin Z. Stanton, Young K. Song, Justin Lack, Chaoyu Wang, Xinyu Wen, Zachary Rae, Adam Cheuk, Gregoire Altan-Bonnet, Michael Kelly, Jun S. Wei, Michael C. Jensen, Rimas J. Orentas, Javed Khan. A comprehensive and integrative omic analysis of multiply relapsed refractory pediatric pre-B cell acute lymphoblastic leukemia predicts response to CD19 CAR T-cell therapy [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A11.