Genomics has transformed the diagnostic landscape of pediatric malignancies by identifying and integrating actionable features that refine diagnosis, classification, and treatment. Yet, translating precision oncology data into effective therapies for hard-to-cure childhood, adolescent, and young adult malignancies remains a significant challenge. We present the case for combining proteomics with patient-derived xenograft models to identify personalized treatment for an adolescent with primary and metastatic spindle epithelial tumor with thymus-like elements (SETTLE). Within two weeks of biopsy, proteomics identified elevated SHMT2 as a target for therapy with the anti-depressant sertraline. Drug response was confirmed within two months using a personalized chicken chorioallantoic membrane model of the patient’s SETTLE tumor. Following failure of cytotoxic chemotherapy and second-line therapy, the patient received sertraline treatment and showed decreased tumor growth rates, albeit with clinically progressive disease. We demonstrate that proteomics and fast-track xenograft models provide supportive pre-clinical data in a clinically meaningful timeframe to impact clinical practice. By this, we show that proteome-guided and functional precision oncology are feasible and valuable complements to the current genome-driven precision oncology practices.

Abstract Translation of precision oncology data into feasible precision therapies for hard-to-cure childhood, adolescent and young adult malignancies remains a significant challenge. Identifying therapeutic targets at the protein and pathway level and demonstrating treatment response in personalized models hold great promise, particularly for combination therapies, but may be considered complex and time consuming. Here, we present the case of an adolescent with metastatic, progressive spindle epithelial tumor with thymus-like differentiation (SETTLE) and evaluate how proteomics combined with rapid patient-derived models can identify treatment options not apparent at the genome or transcript level. Mass spectrometric proteome analysis of macro-dissected tumor and adjacent normal from formalin fixed paraffin embedded sections was completed within two weeks of biopsy and identified key proteins involved in one-carbon metabolism, including SHMT2 and DHFR as possible targets for single or combination therapy. Elevated SHMT2 levels were validated by immunohistochemistry and compared to levels across AYA tumors. Based on the suitability for an innovative therapy trial, we prioritized single-agent sertraline, a commercially available anti-depressant medication that inhibits SHMT2, and confirmed a positive drug response in both chicken chorioallantoic membrane (CAM) and larval zebrafish xenografts generated from the patient. Retrospective expansion in a murine xenograft enabled metabolic tracing on isolated SETTLE- patient-derived xenograft cells using 13C6-glucose confirming SHMT2 activity and response to in vitro treatment. Following failure of cytotoxic chemotherapy and second-line sorafenib treatment, a monotherapy trial of sertraline was initiated by the patient but stopped after 8 weeks after evidence of progressive disease. Possible combination therapies were evaluated further in the patient-derived models. Combining sertraline with the common antibiotic trimethoprim, resulted in enhanced growth inhibition of SETTLE cells in the larval zebrafish xenografts. Significance: Overall, we demonstrate that proteomics and personalized xenograft models may provide supportive pre-clinical data in a clinically meaningful timeframe to support medical decision-making and impact clinical practice. Citation Format: Georgina D. Barnabas, Tariq A. Bhat, Verena Goebeler, Pascal Leclair, Nadine Azzam, Nicole Melong, Jason N. Berman, Jennifer A. Chan, Donna L. Senger, Seth Parker, Christopher A. Maxwell, Gregor S. Reid, Jonathan Bush, Caron Strahlendorf, Rebecca Deyell, C James Lim, Philipp F. Lange. Prioritizing treatment targets for an adolescent with metastatic processive malignancy using proteomics and personalized xenograft models within an actionable timeframe [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 937.

Relapsed precursor B-cell ALL (B-ALL) commonly evolves from minor diagnostic clones, but their early phenotypic characterization remains unachieved despite abundant multi-omics data. Immunophenotyping, an essential diagnostic and therapy-response monitoring tool, identifies the patient-specific expression pattern of bulk leukemia. At time of relapse, most B-ALL immunophenotypes have undergone substantial and seemingly random multi-directional modulations of their diagnostic expression pattern. For decades, these unpredictable immunophenotypic shifts at time of relapse, observed across different methods of antigen expression assessment, have prevented a deeper understanding of phenotypically-defined leukemia progression biology. Addressing this limitation, we applied unsupervised high-dimensional computational analysis of clinical-grade flow cytometry to dissect the intra-leukemic phenotypic heterogeneity at the single-cell level in longitudinally collected B-ALL patients and matched patient-derived xenografts. Our results provide AI-guided and clinically validated evidence that the observed immunophenotypic shifts during disease progression did not result from antigen expression fluctuations, but from enrichment of distinct phenotypically stable subpopulations. As our study identifies patient-specific subpopulation dynamics during disease evolution, it achieves immunophenotypically-defined leukemia progression assessment, which addresses an important unmet clinical and translational need. In each progression series, population dynamics followed a trajectory towards relapse-dominating subpopulations when selective pressures, such as xenotransplantation or in vivo chemotherapy, were applied, often from very minor abundance levels at diagnosis. Each time, the changes in relative proportions of subpopulations explained the observed immunophenotypic shift at the bulk-level. Overcoming decades-old challenges, our findings provide a new conceptual approach to investigate the role of intra-leukemic phenotypic heterogeneity in B-ALL progression to identify treatment-refractory phenotypes, which could significantly impact patient-care, inform precision-medicine options, and enhance relapse-modelling.

Increased access to high-throughput DNA sequencing platforms has transformed the diagnostic landscape of pediatric malignancies by identifying and integrating actionable genomic or transcriptional features that refine diagnosis, classification, and treatment. Yet less than 10% of treated patients show a positive response and translating precision oncology data into feasible and effective therapies for hard-to-cure childhood, adolescent, and young adult malignancies remains a significant challenge. Combining the identification of therapeutic targets at the protein and pathway levels with demonstration of treatment response in personalized models holds great promise. Here we present the case for combining proteomics with patient-derived xenograft (PDX) models to identify personalized treatment options that were not apparent at genomic and transcriptomic levels. Proteome analysis with immunohistochemistry (IHC) validation of formalin-fixed paraffin-embedded sections from an adolescent with primary and metastatic spindle epithelial tumor with thymus-like elements (SETTLE) was completed within two weeks of biopsy. The results identified an elevated protein level of SHMT2 as a possible target for therapy with the commercially available anti-depressant sertraline. Within 2 months and ahead of a molecular tumor board, we confirmed a positive drug response in a personalized chick chorioallantoic membrane (CAM) model of the SETTLE tumor (CAM-PDX). Following the failure of cytotoxic chemotherapy and second-line therapy, a treatment of sertraline was initiated for the patient. After 3 months of sertraline treatment the patient showed decreased tumor growth rates, albeit with clinically progressive disease. Significance: Overall, we demonstrate that proteomics and fast-track personalized xenograft models can provide supportive pre-clinical data in a clinically meaningful timeframe to support medical decision-making and impact the clinical practice. By this we show that proteome-guided and functional precision oncology are feasible and valuable complements to the current genome- driven precision oncology practices. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement Data and/or materials used for this research were made available by the PRecision Oncology For Young peopLE (PROFYLE) program. PROFYLE has been supported by funds from many funders including Alberta Cancer Foundation, Alberta Childrens Hospital Foundation, BC Cancer Foundation, BC Childrens Hospital Foundation, Childhood Cancer Canada, Kids Cancer Care Foundation and Terry Fox Research Institute. This work was supported by the BC Childrens Hospital Foundation through the Better Responses through Avatars and Evidence (BRAvE) Initiative. Salary support was provided by the Michael Cuccione Foundation MCF (C.J.L., G.S.D.R., C.A.M., P.F.L., V.G.), the Canada Research Chairs Program (CRC-RS 950-230867, P.F.L.), the Canadian Institutes of Health Research (C.A.M., P.F.L.), the Michael Smith Foundation for Health Research Scholar Program (16442, P.F.L.), MITACS (T.A.B., G.B.) and the University of British Columbia (E.K.E.). Project support for J.A.C. and D.L.S. was provided by The Alberta Cancer Foundation. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The Children and Womens Research Ethics Board of the University of British Columbia gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes To maintain the patients and familys privacy in this study of a single case the raw data is not made available at this point. Release of the raw data as part of an aggregated patient cohort is in preparation.

Abstract Molecularly targeted precision treatments have significant potential to improve therapy options for hard-to-treat cancers and reduce late effects in general. Genome sequencing has laid the foundation for precision medicine, yet, clinical success remains moderate. Identifying therapeutic targets at the protein and pathway level holds great promise, but may be considered complex. This is particularly true for smaller centres without local proteomics expertise, raising concern of new access barriers and increased inequity. Here, we present a road map for the nation-wide integration of pre-clinical proteomics evidence into pediatric precision oncology and provide proof of concept for proteome guided therapies. Within the Canadian Pediatric Cancer Consortium - ACCESS (Advancing Childhood Cancer Experience, Science & Survivorship) and the PROFYLE study (Precision Oncology for Young peopLE) we establish a federated proteomics platform and a National Molecular Pathology Board to promote equitable access to proteomics analyses and other innovative but not universally available molecular tests. As proof of concept, we describe the case of an adolescent with metastatic, progressive spindle epithelial tumor with thymus-like differentiation (SETTLE) and evaluate how quantitative proteome profiling can identify treatment options not apparent at the genome or transcript level. Mass spectrometric proteome analysis of macro-dissected tumor and adjacent normal from formalin fixed paraffin embedded sections was completed within two weeks of biopsy and identified key proteins involved in one-carbon metabolism, including SHMT2 and DHFR as possible targets for single or combination therapy. SHMT2 levels were validated and compared across various tumors by immunohistochemistry and the response to SHMT2 inhibition by sertraline was tested in chicken chorioallantoic membrane (CAM) and larval zebrafish xenografts generated from the patient. Following failure of cytotoxic chemotherapy and second-line sorafenib treatment, a monotherapy trial was initiated by the patient. Treatment was stopped after 8 weeks following evidence of a moderate reduction in growth rate but overall progressive disease. Possible combination therapies were evaluated further in the patient-derived models. Significance: Overall, we demonstrate that pre-clinical proteomics analysis and validation can be conducted in a clinically meaningful timeframe and has provided supportive pre-clinical data to support medical decision-making in one case of a rare progressive malignancy. To determine if this is generalizable, we further outline a road map for advancing access to pre-clinical proteomics and innovative molecular assays across Canada. Citation Format: Georgina Barnabas, Tariq Bhat, V. Goebeler, P. Leclair, C. Maxwell, G. Reid, D. L. Senger, J. A. Chan, N. Azzam, N. Melong, J. N. Berman, S Parker, J. Bush, C. Strahlendorf, R. Deyell, C. J. Lim, PROFYLE Program, Philipp F. Lange. Canada’s path towards proteome guided therapies and advanced molecular pathology in pediatric precision oncology [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pediatric Cancer Research; 2024 Sep 5-8; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl):Abstract nr PR003.

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Of the four molecular subgroups, Group 3 MB is the most aggressive and has the worst prognosis. To understand the origins of chemoresistance involving IL-6/STAT3 signaling, we used in vitro co-culture systems to investigate the contribution of microglia as a brain tumor microenvironment cellular source of paracrine cytokines that promotes acquired drug resistance in Group 3 MB. MB cells subjected to co-culture with microglia exhibited increased expression of phosphorylated JAK1 and STAT3, which was correlated with enhanced resistance to vincristine. We found that both microglia and MB cells co-cultured with microglia secreted significant quantities of IL-6, indicating that IL-6 is a paracrine and autocrine cytokine able to initiate and sustain STAT3 activity in MB cells. Surprisingly, IL-6R−/− MB cells, which cannot respond to exogenous IL-6 stimuli, were responsive to microglia co-culture induced activation of STAT3 and chemoresistance. Subsequently, we found that MB cells conditioned in vitro with the IL-6 family cytokines, IL-6, OSM, LIF, or IL-11, exhibited enhanced JAK1/STAT3 activity and chemoresistance. Intriguingly, MB cells conditioned with any one of the IL-6 family cytokine secreted multiple IL-6 family cytokines, implicating a feedback network involving multiple cytokines. The IL-6 family cytokine receptors share a common signal transducing β-subunit, gp130, which may be targeted to mitigate tumor chemoresistance. We showed that microglia co-culture failed to induce chemoresistance of gp130−/− MB cells, and that combination treatment using gp130 inhibitors, or with the JAK inhibitor ruxolitinib, effectively overcame the observed resistance to vincristine in gp130 expressing MB cells. Our in vitro studies highlight the gp130/JAK/STAT pathway as a therapeutic target in combating acquired treatment resistance in Group 3 MB.

Abstract Medulloblastoma (MB) is the most common pediatric brain tumour comprising of four distinct molecular subgroups exhibiting high level of intertumoural heterogeneity. The standard multimodal treatment yields a survival rate of 70% but the aggressive treatment affects the long-term sequelae of MB patients. The tumour microenvironment (TME) is a regulator of cancer progression and affects therapeutic efficacy in primary and metastatic brain malignancies. Mechanistic insights into the tumor-promoting role of the individual components of the brain TME will aid in identifying key survival pathways and design of potential therapeutics to combat drug resistance and pathogenesis of the disease. We hypothesize that tumor associated macrophages (TAMs) in the brain TME facilitate drug resistance via the IL-6/STAT3 signalling axis in MB. To understand the role of paracrine signaling resulting from TAMs, a co-culture system was used to evaluate the expression of pSTAT3 and correlated with drug sensitivity and/or resistance in Med8A cell line. When co-cultured with HMC3, a human microglia cell line, the chemosensitive Med8A-S cells exhibited enhanced pSTAT3 expression and acquired drug resistance. Interestingly, Med8A cells lacking expression of IL-6 receptor (IL6R-/-) also exhibited high pSTAT3 levels and drug resistance; suggesting that HMC3 may release soluble factors in addition to IL-6 sufficient to drive chemoresistance in Med8A variants. To assess this, we conditioned cells with stimulatory cytokines belonging to the IL-6 family. We found that oncostatin M (OSM), interleukin-11 (IL-11) and leukemia inhibitory factor (LIF) induced drug resistance and enhanced pSTAT3 levels in IL6R-/- cells. Receptors of the IL-6 family share a common signal transducing beta subunit, glycoprotein (gp130) that transduces the signal intracellularly and phosphorylates janus activated kinases (JAKs) and subsequently STAT3. Our results suggest that paracrine proinflammatory cytokines found in the TME can promote MB drug resistance by signalling through a common signal transducer gp130, hence targeting any one cytokine receptor of the IL-6 family of cytokines may not sufficiently abrogate acquired drug resistance. Currently, we are evaluating the efficacy of agents that target common downstream elements of the IL-6/STAT3 signalling axis, including gp130, JAKs and STAT3, as a potentially improved therapeutic strategy to circumvent acquired drug resistance in medulloblastoma. Citation Format: Lakshana Sreenivasan, Pascal Leclair, Chinten James Lim. Targetting IL-6/gp130 signalling axis attenuates acquired drug resistance in medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB133.

Human immunodeficiency virus (HIV) remodels the cell surface of infected cells to facilitate viral dissemination and promote immune evasion. The membrane-associated Vpu accessory protein encoded by HIV-1 plays a key role in this process by altering cell surface levels of multiple host proteins. Using an unbiased quantitative plasma membrane profiling approach, we previously identified CD47 as a putative host target downregulated by Vpu. CD47 is a ubiquitously-expressed cell surface protein that interacts with the myeloid cell inhibitory receptor SIRPα to deliver a “don’t-eat-me” signal, thus protecting cells from phagocytosis. In this study, we investigate whether CD47 modulation by HIV-1 Vpu might promote the susceptibility of macrophages to viral infection via phagocytosis of infected CD4+ T cells. Indeed, we find that Vpu downregulates CD47 expression on infected CD4+ T cells leading to an enhanced capture and phagocytosis by macrophages. Interestingly, it is through this process that a CCR5-tropic transmitted/founder (T/F) virus, which otherwise poorly infects macrophages in its cell-free form, becomes infectious in macrophages. Importantly, we show that HIV-1-infected cells expressing a Vpu-resistant CD47 mutant are less prone to infect macrophages through phagocytosis. Mechanistically, Vpu forms a physical complex with CD47 through its transmembrane domain and targets the latter for lysosomal degradation. These results reveal a novel role of Vpu in modulating macrophage infection, which has important implications for HIV-1 transmission in early stages of infection and the establishment of viral reservoir.IMPORTANCE Macrophages play critical roles in HIV transmission, viral spread early in infection, and as a reservoir of virus. Selective capture and engulfment of HIV-1 infected T cells was shown to drive efficient macrophage infection suggesting that this mechanism represents an important mode of infection notably for weakly macrophage-tropic T/F viruses. In this study, we provide insight into the signals that regulate this process. We show that the HIV-1 accessory protein Vpu downregulates cell surface levels of CD47, a host protein that interacts with the inhibitory receptor SIRPα to deliver a “don’t-eat-me” signal to macrophages. This allows for enhanced capture and phagocytosis of infected T cells by macrophages, ultimately leading to their productive infection even with T/F virus. These findings provide new insights into the mechanisms governing the intercellular transmission of HIV-1 to macrophages with implications for the establishment of the macrophage reservoir and early HIV-1 dissemination in vivo .

Medulloblastoma (MB) is the most common pediatric brain tumour comprising of four distinct molecular subgroups exhibiting high level of intertumoural heterogeneity. The standard multimodal treatment yields a survival rate of 70% but the aggressive treatment affects the long-term sequelae of MB patients. The tumour microenvironment (TME) is a regulator of cancer progression and affects therapeutic efficacy in primary and metastatic brain malignancies. Mechanistic insights into the tumor-promoting role of the individual components of the brain TME will aid in identifying key survival pathways and design of potential therapeutics to combat drug resistance and pathogenesis of the disease. We hypothesize that tumor associated macrophages (TAMs) in the brain TME facilitate drug resistance via the IL-6/STAT3 signalling axis in MB. To understand the role of paracrine signaling resulting from TAMs, a co-culture system was used to evaluate the expression of pSTAT3 and correlated with drug sensitivity and/or resistance in Med8A cell line. When co-cultured with HMC3, a human microglia cell line, the chemosensitive Med8A-S cells exhibited enhanced pSTAT3 expression and acquired drug resistance. Interestingly, Med8A cells lacking expression of IL-6 receptor (IL6R-/-) also exhibited high pSTAT3 levels and drug resistance; suggesting that HMC3 may release soluble factors in addition to IL-6 sufficient to drive chemoresistance in Med8A variants. To assess this, we conditioned cells with stimulatory cytokines belonging to the IL-6 family. We found that oncostatin M (OSM), interleukin-11 (IL-11) and leukemia inhibitory factor (LIF) induced drug resistance and enhanced pSTAT3 levels in IL6R-/- cells. Receptors of the IL-6 family share a common signal transducing beta subunit, glycoprotein (gp130) that transduces the signal intracellularly and phosphorylates janus activated kinases (JAKs) and subsequently STAT3. Our results suggest that paracrine proinflammatory cytokines found in the TME can promote MB drug resistance by signalling through a common signal transducer gp130, hence targeting any one cytokine receptor of the IL-6 family of cytokines may not sufficiently abrogate acquired drug resistance. Currently, we are evaluating the efficacy of agents that target common downstream elements of the IL-6/STAT3 signalling axis, including gp130, JAKs and STAT3, as a potentially improved therapeutic strategy to circumvent acquired drug resistance in medulloblastoma. Citation Format: Lakshana Sreenivasan, Pascal Leclair, Chinten James Lim. Targetting IL-6/gp130 signalling axis attenuates acquired drug resistance in medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB133.

Genome-wide association studies have shown that a gene variant in the Family with sequence similarity 13, member A (FAM13A) is strongly associated with reduced lung function and the appearance of respiratory symptoms in patients with chronic obstructive pulmonary disease (COPD). A key player in smoking-induced tissue injury and airway remodeling is the transforming growth factor-β1 (TGF-β1). To determine the role of FAM13A in TGF-β1 signaling, FAM13A-/- airway epithelial cells were generated using CRISPR-Cas9, whereas overexpression of FAM13A was achieved using lipid nanoparticles. Wild-type (WT) and FAM13A-/- cells were treated with TGF-β1, followed by gene and/or protein expression analyses. FAM13A-/- cells augmented TGF-β1-induced increase in collagen type 1 (COL1A1), matrix metalloproteinase 2 (MMP2), expression compared with WT cells. This effect was mediated by an increase in β-catenin (CTNNB1) expression in FAM13A-/- cells compared with WT cells after TGF-β1 treatment. FAM13A overexpression was partially protective from TGF-β1-induced COL1A1 expression. Finally, we showed that airway epithelial-specific FAM13A protein expression is significantly increased in patients with severe COPD compared with control nonsmokers, and negatively correlated with lung function. In contrast, β-catenin (CTNNB1), which has previously been linked to be regulated by FAM13A, is decreased in the airway epithelium of smokers with COPD compared with non-COPD subjects. Together, our data showed that FAM13A may be protective from TGF-β1-induced fibrotic response in the airway epithelium via sequestering CTNNB1 from its regulation on downstream targets. Therapeutic increase in FAM13A expression in the airway epithelium of smokers at risk for COPD, and those with mild COPD, may reduce the extent of airway tissue remodeling.

CD47 is a tumor-associated antigen best known for its ability to bind counter-receptors on the surface of professional phagocytes as an immune-evasion strategy. ⁣Recently, CD47 has been shown to play a role as a signaling receptor, involving a number of cell physiological processes.⁣⁣ This review provides a comprehensive survey of the signaling pathways triggered by CD47 ligand-mediated cell death in tumor cells. Such an understanding should lead to improvement of CD47-targeted anti-tumor therapeutics able to both neutralize the anti-phagocytic role and trigger autonomous tumor cell death.

Medulloblastoma (MB) is a high-grade pediatric brain malignancy that originates from neuronal precursors located in the posterior cranial fossa. In this study, we evaluated the role of STAT3 and IL-6 in a tumor microenvironment mediated drug resistance in human MBs. We established that the Group 3 MB cell line, Med8A, is chemosensitive (hence Med8A-S), and this is correlated with a basal low phosphorylated state of STAT3, while treatment with IL-6 induced robust increases in pY705-STAT3. Via incremental selection with vincristine, we derived the stably chemoresistant variant, Med8A-R, that exhibited multi-drug resistance, enhanced IL-6 induced pY705-STAT3 levels, and increased IL6R expression. Consequently, abrogation of STAT3 or IL6R expression in Med8A-R led to restored chemosensitivity to vincristine, highlighting a prominent role for canonical IL-6/STAT3 signaling in acquired drug resistance. Furthermore, Med8A-S subjected to conditioning exposure with IL-6, termed Med8A-IL6+ cells, exhibited enhanced vincristine resistance, increased expression of pY705-STAT3 and IL6R, and increased secretion of IL-6. When cocultured with Med8A-IL6+ cells, Med8A-S cells exhibited increased pY705-STAT3 and increased IL-6 secretion, suggesting a cytokine feedback loop responsible for amplifying STAT3 activity. Similar IL-6 induced phenomena were also observed in the Group 3 MB cell lines, D283 and D341, including increased pY705-STAT3, drug resistance, IL-6 secretion and IL6R expression. Our study unveiled autocrine IL-6 as a promoter of STAT3 signaling in development of drug resistance, and suggests therapeutic benefits for targeting the IL-6/STAT3 signaling axis in Group 3 MBs.

Despite complete remissions being achieved in most newly diagnosed acute lymphoblastic leukemia (ALL) patients, relapse remains a significant clinical challenge. Interrogation of matched ALL samples has provided insights into the cellular characteristics and evolutionary trajectory of relapse-fated clones, but requires blasts obtained at time of relapse to provide a reference point for the retrospective analysis. Despite the strong prognostic value of minimal residual disease (MRD), MRD-positive samples have been under-utilized for investigating disease progression as the low leukemia burden presents significant challenges to the evaluation of the residual blasts. To overcome this limitation, we performed patient-derived xenografting (PDX) with MRD samples to enable deeper characterization of residual blasts, including functional assessment of their leukemia propagating ability. In marked contrast to a previous study in adult ALL, we achieved engraftment of MRD blasts from 5 of 7 pediatric ALL patient samples, obtained at the end of induction (EOI) therapy, by intravenous injection into non-conditioned NSG mice. MRD blasts expanded with patient-specific kinetics, reaching overt leukemia in recipients of three of the samples and remaining at MRD levels in recipients of the remaining two. The absence of normal human hematopoiesis in the MRD-engrafted mice (termed EOI-PDX), combined with the expansion of residual blast numbers, enabled clear phenotypic definition of therapy-selected, leukemia-propagating subpopulations, including those present at <1%. Subsequent reanalysis of diagnosis (DX) and EOI clinical samples for the identified subpopulations exposed a treatment response-mediated disruption of the established population hierarchy that explained the phenotypic shifts observed during disease progression. The relevance of the subpopulations identified as potential leukemia drivers was confirmed by their dominance in subsequent clinical relapses. Furthermore, phenotype-based sorting of viable blast subpopulations enabled the identification and comparison of metabolic characteristics of treatment sensitive and resistant blasts. This immunophenotypic study recapitulates the enrichment of minor clones during ALL progression described by retrospective sequencing studies, but it does so prospectively. By expanding on the quantitative and qualitative information that can be gathered from MRD samples, EOI-PDX identify the blast populations that are most likely to drive relapse, prior to clinical disease progression. By providing a platform to achieve early identification and ongoing monitoring of therapy-resistant subpopulations with strong leukemia-propagating capacity, this approach could refine treatment-response assessment and relapse-risk stratification. Further, by expanding the numbers of viable treatment-selected MRD blasts available for interrogation, EOI-PDX could enable early identification of vulnerabilities in chemotherapy-resistant blasts. Disclosures No relevant conflicts of interest to declare.

Dear Editor, Uscanga-Palomeque et al reported that PKHB1, a peptide analog of 4N1K with increased stability in serum, directly induced T-leukemic cell death by engaging the CD47 receptor.1 CD47 is commonly upregulated in tumor cells, and its binding to macrophage SIRPα inhibits phagocytosis and engagement of the innate immune response.2 It is well documented that CD47 ligation with antibodies can invoke cell death in the absence of effector cells.3, 4 The potential to convert CD47 from a protumor antigen to one that is antitumor has attracted the search for CD47-targeted therapeutics. In this vein, mapping studies of thrombospondin, a natural ligand of CD47, led to discovery of the VVM-motif containing peptides, 4N1 and 7N3, that mediate binding to the CD47 IgV domain. To improve solubility, N/C-terminal lysines were added to yield the peptide known as 4N1K, with the sequence KRFYVVMWKK.5 PKHB1 has an identical sequence, but with terminal D-lysines instead of the L-enantiomer found in 4N1K.6 It was reported in 2001 that 4N1K mediated significant biological effects that were CD47-independent.7, 8 In 2014, we published a study that highlighted the propensity of 4N1K to bind nonspecifically to proteins in vitro as well as to the plasma membrane, and that this phenomenon was a major contributor to the cellular effects mediated by 4N1K.9 This included the apparent “induction” of integrin activation that was presumed to occur through CD47 ligation by 4N1K. We showed that 4N1K treatment promoted binding of several antibodies to Jurkat T-leukemic cells, including the binding of β1-integrin specific antibodies to a Jurkat derivative that expressed no β1-integrins.9 We obtained similar results using only secondary antibodies, leading us to posit that 4N1K mediated CD47-independent binding to a variety of Ig domains on the cell surface. Indeed, we used cell-free assays to show that different antibodies readily interacted with 4N1K that was immobilized on plastic. Thus, we concluded that 4N1K had significant CD47-independent effects and, as such, any experiment using this and similar peptides should be accompanied by stringent negative controls, such as CD47 knockdown or knockout cells. Another group working on microglia biology has since corroborated our results on 4N1K.10 Given that 4N1K is known to have substantial CD47-independent effects, it is conceivable that PKHB1 may as well. One aspect we had not investigated previously was CD47-mediated cell death.9 As such, we evaluated WT and CD47−/− Jurkat and MOLT4 cell lines to evaluate the ability of PKHB1 and 4N1K to induce T-leukemic cell death. Here, we report that both PKHB1 and 4N1K induced significant cell death in a CD47-independent manner, ascertained in two ways: (i) the forward and side scatter profile of WT and CD47−/− cells treated with PKHB1 or 4N1K was similar, presenting a population with decreased size and increased granularity, characteristic of cells undergoing cell death (Figure 1A); and (ii) there was significant annexin V binding when CD47−/− cells were treated with 4N1K or PKHB1 (Figure 1B). In contrast, and as previously reported,4 the anti-CD47 antibody CC2C6 induced cell death in WT, but not in CD47−/−, Jurkat cells. We note with interest that MOLT4 cells were not sensitive to CC2C6-CD47 ligation-induced death, but more importantly, both WT and CD47−/− MOLT4 cells exhibit equal susceptibility to PKHB1 or 4N1K treatment-induced death (Figure 1B). Given that the CD47-independent effects of PKHB1 were likely to be similar to those of 4N1K, we repeated select key experiments performed previously9 to shed light on the mechanism of PKHB1-mediated, but CD47-independent, effects. Wild-type and CD47−/− cells treated with or without peptides were incubated with an anti-CD47 antibody, B6H12, followed by a fluorophore-conjugated secondary antibody. As shown in Figure 2A, both CD47−/− cells treated with PKHB1 or 4N1K exhibited significant labeling with B6H12. The non-CD47-specific labeling is directly attributed to nonspecific antibody binding to PKHB1 or 4N1K treated cells, as evident from positively labeled cells incubated with only secondary antibodies (Figure 2A). Importantly, this phenomenon is peptide dose-dependent, as demonstrated by increased binding of CD47−/− cells with increasing concentrations of PKHB1 or 4N1K, but not of 4NGG (Figure 2B). Previously, we showed, using a simple cell-free assay, that 4N1K immobilized on plastic effectively bound a fluorophore-conjugated secondary antibody.9 We repeated this assay to include PKHB1, and found that, similar to 4N1K, PKHB1 also bound to a secondary antibody in a dose-dependent manner (Figure 2C). Our results essentially agree with those of Uscanga-Palomeque et al1 in that PKHB1 and 4N1K are peptides that appear to induce leukemic cell death in an efficient and rapid manner, similar to what has been reported for certain CD47 antibodies.3, 4 However, the peptides clearly function in a manner that does not involve CD47 as a receptor, a definitive result based on the use of two CD47−/− leukemic cell lines as the required and critical controls. We reiterate exercising caution when interpreting 4N1K- or PKHB1-induced cell phenomena; the accumulated evidence clearly indicates that the effects have no bearing on CD47 as the presumptive receptor, and calls into question the further development of these peptides and other derivatives as CD47-targeted therapeutics. The authors have no conflict of interest.

Therapy-induced presentation of cell surface calreticulin (CRT) is a pro-phagocytic immunogen beneficial for invoking anti-tumor immunity. Here, we characterized the roles of ERp57 and α-integrins as CRT-interacting proteins that coordinately regulate CRT translocation from the ER to the surface during immunogenic cell death. Using T-lymphoblasts as a genetic cell model, we found that drug-induced surface CRT is dependent on ERp57, while drug-induced surface ERp57 is independent of CRT. Differential subcellular immunostaining assays revealed that ERp57-/- cells have minimal cytosolic CRT, indicating that ERp57 is indispensable for extra-ER accumulation of CRT. Stimulation of integrin activity, with either cell adhesion or molecular agonists, resulted in decreased drug-induced surface CRT and ERp57 levels. Similarly, surface CRT and ERp57 was reduced in cells expressing GFFKR, a conserved α-integrin cytosolic motif that binds CRT. Drug-induced surface ERp57 levels were consistently higher in CRT-/- cells, suggesting integrin inhibition of surface ERp57 is an indirect consequence of α-integrin binding to CRT within the CRT-ERp57 complex. Furthermore, β1-/- cells with reduced expression of multiple α-integrins, exhibit enhanced levels of drug-induced surface CRT and ERp57. Our findings highlight the coordinate involvement of plasma membrane integrins as inhibitors, and ERp57 originating from the ER as promoters, of CRT translocation from the ER to the cell surface.