The BIM deletion polymorphism potentiates the survival of leukemia stem and progenitor cells and impairs response to targeted therapies.

One sixth of human cancers harbor pathogenic germline variants, but few studies have established their functional contribution to cancer outcomes. Here, we developed a humanized mouse model harboring a common East Asian polymorphism, the BIM deletion polymorphism (BDP), which confers resistance to oncogenic kinase inhibitors through generation of non-apoptotic splice isoforms. However, despite its clear role in mediating bulk resistance in patients, the BDP's role in cancer stem and progenitor cells, which initiate disease and possess altered BCL-2 rheostats compared to differentiated tumor cells, remains unknown. To study the role of the BDP in leukemia initiation, we crossed the BDP mouse into a chronic myeloid leukemia (CML) model. We found that the BDP greatly enhanced the fitness of CML cells with a three-fold greater competitive advantage, leading to more aggressive disease. The BDP conferred almost complete resistance to cell death induced by imatinib in CML stem and progenitor cells (LSPCs). Using BH3 profiling, we identified a novel therapeutic vulnerability of BDP LSPCs to MCL-1 antagonists, which we confirmed in primary human LSPCs, and in vivo. Our findings demonstrate the impact of human polymorphisms on the survival of LSPCs and highlight their potential as companion diagnostics for tailored therapies.

Activation of NOTCH signaling impedes cell proliferation and survival in acute megakaryoblastic leukemia.

The genetic lesions that drive acute megakaryoblastic leukemia (AMKL) have not been fully elucidated. To search for genetic alterations in AMKL, we performed targeted deep sequencing in 34 AMKL patient samples and 8 AMKL cell lines and detected frequent genetic mutations in the NOTCH pathway in addition to previously reported alterations in GATA-1 and the JAK-STAT pathway. Pharmacological and genetic NOTCH activation, but not inhibition, significantly suppressed AMKL cell proliferation in both in vitro and in vivo assays employing a patient-derived xenograft model. These results suggest that NOTCH inactivation underlies AMKL leukemogenesis. and NOTCH activation holds the potential for therapeutic application in AMKL.

A phase I/II study of adoptive SARS-CoV-2-specific T cells in immunocompromised hosts with or at risk of severe COVID-19 infection.

BACKGROUND: Post-transplant or hematological cancer patients have a higher risk of mortality after infection with ancestral and early variants of severe acute respiratory syndrome (SARS)-CoV-2. Adoptive cell therapy (ACT) with virus-specific T cells (VSTs) could augment endogenous T cell immunity to avoid disease deterioration before viral clearance. METHODS: We established a third-party SARS-CoV-2-specific T cell (COVID-T) bank in 2020 (NCT04351659) using convalescent and/or vaccinated donors. In a phase I/II study (NCT04457726), 13 adult and pediatric patients, acutely positive for SARS-CoV-2 and predicted to have a high chance of mortality, were recruited from September 2021 to February 2022. Twelve patients received a single dose of COVID-T cells, matched on at least 1 HLA. RESULTS: A dose of either 75,000 or 150,000 IFN-γ+CD3+ cells/m2 SARS-COV-2-specific T cells did not cause cytokine release syndrome, acute respiratory distress syndrome, or graft-versus-host disease. In the 8 patients who had detectable donor SARS-COV-2-specific T cells after ACT, none progressed to severe disease or died with COVID-19. In contrast, among the other four patients without evidence of donor micro-chimerism, two died of COVID-19. CONCLUSIONS: Long-acting third-party VSTs from convalescent or vaccinated donors could be expediently produced and might be clinically useful in future pandemics, particularly before global vaccination is implemented.

Rapid production of clinical-grade SARS-CoV-2 specific T cells.

Objectives: To determine whether the frequencies of SARS-CoV-2-specific T cells are sufficiently high in the blood of convalescent donors and whether it is technically feasible to manufacture clinical-grade products overnight for T-cell therapy and assessment of COVID-19 immunity. Methods: One unit of whole blood or leukapheresis was collected from each donor following standard blood bank practices. The leukocytes were stimulated using overlapping peptides of SARS-CoV-2, covering the immunodominant sequence domains of the S protein and the complete sequence of the N and M proteins. Thereafter, functionally reactive cells were enriched overnight using an automated device capturing IFNγ-secreting cells. Results: From 1 × 109 leukocytes, a median of 0.98 × 106 (range 0.56-2.95) IFNγ + T cells were produced from each of the six donors, suggesting a high frequency of SARS-CoV-2 reactive T cells in their blood, even though only one donor had severe COVID-19 requiring mechanical ventilation whereas the other five donors had minor symptoms. A median of 57% of the enriched T cells were IFNγ+ (range 20%-74%), with preferential enrichment of CD56+ T cells and effector memory T cells. TCRVß-spectratyping confirmed distinctively tall oligoclonal peaks in final products. With just six donors, the probability that a recipient would share at least one HLA allele with one of the donors is >88% among Caucasian, >95% among Chinese, >97% among Malay, and >99% among Indian populations. Conclusions: High frequencies of rapid antigen-reactive T cells were found in convalescent donors, regardless of severity of COVID-19. The feasibility of clinical-grade production of SARS-CoV-2-specific T cells overnight for therapeutics and diagnostics is revealed.

An integrative model of pathway convergence in genetically heterogeneous blast crisis chronic myeloid leukemia.

Targeted therapies against the BCR-ABL1 kinase have revolutionized treatment of chronic phase (CP) chronic myeloid leukemia (CML). In contrast, management of blast crisis (BC) CML remains challenging because BC cells acquire complex molecular alterations that confer stemness features to progenitor populations and resistance to BCR-ABL1 tyrosine kinase inhibitors. Comprehensive models of BC transformation have proved elusive because of the rarity and genetic heterogeneity of BC, but are important for developing biomarkers predicting BC progression and effective therapies. To better understand BC, we performed an integrated multiomics analysis of 74 CP and BC samples using whole-genome and exome sequencing, transcriptome and methylome profiling, and chromatin immunoprecipitation followed by high-throughput sequencing. Employing pathway-based analysis, we found the BC genome was significantly enriched for mutations affecting components of the polycomb repressive complex (PRC) pathway. While transcriptomically, BC progenitors were enriched and depleted for PRC1- and PRC2-related gene sets respectively. By integrating our data sets, we determined that BC progenitors undergo PRC-driven epigenetic reprogramming toward a convergent transcriptomic state. Specifically, PRC2 directs BC DNA hypermethylation, which in turn silences key genes involved in myeloid differentiation and tumor suppressor function via so-called epigenetic switching, whereas PRC1 represses an overlapping and distinct set of genes, including novel BC tumor suppressors. On the basis of these observations, we developed an integrated model of BC that facilitated the identification of combinatorial therapies capable of reversing BC reprogramming (decitabine+PRC1 inhibitors), novel PRC-silenced tumor suppressor genes (NR4A2), and gene expression signatures predictive of disease progression and drug resistance in CP.

The arginase inhibitor Nω-hydroxy-nor-arginine (nor-NOHA) induces apoptosis in leukemic cells specifically under hypoxic conditions but CRISPR/Cas9 excludes arginase 2 (ARG2) as the functional target.

Cancer cells, including in chronic myeloid leukemia (CML), depend on the hypoxic response to persist in hosts and evade therapy. Accordingly, there is significant interest in drugging cancer-specific hypoxic responses. However, a major challenge in leukemia is identifying differential and druggable hypoxic responses between leukemic and normal cells. Previously, we found that arginase 2 (ARG2), an enzyme of the urea cycle, is overexpressed in CML but not normal progenitors. ARG2 is a target of the hypoxia inducible factors (HIF1-α and HIF2-α), and is required for the generation of polyamines which are required for cell growth. We therefore explored if the clinically-tested arginase inhibitor Nω-hydroxy-nor-arginine (nor-NOHA) would be effective against leukemic cells under hypoxic conditions. Remarkably, nor-NOHA effectively induced apoptosis in ARG2-expressing cells under hypoxia but not normoxia. Co-treatment with nor-NOHA overcame hypoxia-mediated resistance towards BCR-ABL1 kinase inhibitors. While nor-NOHA itself is promising in targeting the leukemia hypoxic response, we unexpectedly found that its anti-leukemic activity was independent of ARG2 inhibition. Genetic ablation of ARG2 using CRISPR/Cas9 had no effect on the viability of leukemic cells and their sensitivity towards nor-NOHA. This discrepancy was further evidenced by the distinct effects of ARG2 knockouts and nor-NOHA on cellular respiration. In conclusion, we show that nor-NOHA has significant but off-target anti-leukemic activity among ARG2-expressing hypoxic cells. Since nor-NOHA has been employed in clinical trials, and is widely used in studies on endothelial dysfunction, immunosuppression and metabolism, the diverse biological effects of nor-NOHA must be cautiously evaluated before attributing its activity to ARG inhibition.

RGG boxes within the TET/FET family of RNA-binding proteins are functionally distinct.

The multi-functional TET (TAF15/EWS/TLS) or FET (FUS/EWS/TLS) protein family of higher organisms harbor a transcriptional-activation domain (EAD) and an RNA-binding domain (RBD). The transcriptional activation function is, however, only revealed in oncogenic TET-fusion proteins because in native TET proteins it is auto-repressed by RGG-boxes within the TET RBD. Auto-repression is suggested to involve direct cation-pi interactions between multiple Arg residues within RGG boxes and EAD aromatics. Via analysis of TET transcriptional activity in different organisms, we report herein that repression is not autonomous but instead requires additional trans-acting factors. This finding is not supportive of a proposed model whereby repression occurs via a simple intramolecular EAD/RGG-box interaction. We also show that RGG-boxes present within reiterated YGGDRGG repeats that are unique to TAF15, are defective for repression due to the conserved Asp residue. Thus, RGG boxes within TET proteins can be functionally distinguished. While our results show that YGGDRGG repeats are not involved in TAF15 auto-repression, their remarkable number and conservation strongly suggest that they may confer specialized properties to TAF15 and thus contribute to functional differentiation within the TET/FET protein family.

The BIM deletion polymorphism: A paradigm of a permissive interaction between germline and acquired TKI resistance factors in chronic myeloid leukemia.

Both germline polymorphisms and tumor-specific genetic alterations can determine the response of a cancer to a given therapy. We previously reported a germline deletion polymorphism in the BIM gene that was sufficient to mediate intrinsic resistance to tyrosine kinase inhibitors (TKI) in chronic myeloid leukemia (CML), as well as other cancers [1]. The deletion polymorphism favored the generation of BIM splice forms lacking the pro-apoptotic BH3 domain, conferring a relative resistance to the TKI imatinib (IM). However, CML patients with the BIM deletion polymorphism developed both partial and complete IM resistance. To understand the mechanisms underlying the latter, we grew CML cells either with or without the BIM deletion polymorphism in increasing IM concentrations. Under these conditions, the BIM deletion polymorphism enhanced the emergence of populations with complete IM resistance, mimicking the situation in patients. Importantly, the combined use of TKIs with the BH3 mimetic ABT-737 overcame the BCR-ABL1-dependent and -independent resistance mechanisms found in these cells. Our results illustrate the interplay between germline and acquired genetic factors in confering TKI resistance, and suggest a therapeutic strategy for patients with complete TKI resistance associated with the BIM deletion polymorphism.

The BCL2 inhibitor ABT-199 significantly enhances imatinib-induced cell death in chronic myeloid leukemia progenitors.

BCR-ABL1-specific tyrosine kinase inhibitors prolong the life of patients with chronic myeloid leukemia (CML) but cannot completely eradicate CML progenitors. The BH3 mimetic, ABT-263, targets prosurvival BCL2 family members, and has activity against CML progenitors. However, the inhibitory effect of ABT-263 on BCL-XL, which mediates platelet survival, produces dose-limiting thrombocytopenia. A second-generation BH3 mimetic, ABT-199, has been developed to specifically bind BCL2 but not BCL-XL. We determined the activity of ABT-199 against CML cell lines, as well as primary CML and normal cord blood (NCB) progenitors. We find that BCL2 expression levels predict sensitivity to ABT-199 in CML and NCB progenitors, and that high NCB BCL2 levels may explain the reported hematologic toxicities in ABT-199-treated patients. Also, while single agent ABT-199 has modest activity against CML progenitors, when combined with imatinib, ABT-199 significantly enhances imatinib activity against CML progenitors at concentrations predicted to avoid hematologic toxicities.

Physiologic hypoxia promotes maintenance of CML stem cells despite effective BCR-ABL1 inhibition.

C-abl oncogene 1, nonreceptor tyrosine kinase (ABL1) kinase inhibitors such as imatinib mesylate (imatinib) are effective in managing chronic myeloid leukemia (CML) but incapable of eliminating leukemia stem cells (LSCs), suggesting that kinase-independent pathways support LSC survival. Given that the bone marrow (BM) hypoxic microenvironment supports hematopoietic stem cells, we investigated whether hypoxia similarly contributes to LSC persistence. Importantly, we found that although breakpoint cluster region (BCR)-ABL1 kinase remained effectively inhibited by imatinib under hypoxia, apoptosis became partially suppressed. Furthermore, hypoxia enhanced the clonogenicity of CML cells, as well as their efficiency in repopulating immunodeficient mice, both in the presence and absence of imatinib. Hypoxia-inducible factor 1 α (HIF1-α), which is the master regulator of the hypoxia transcriptional response, is expressed in the BM specimens of CML individuals. In vitro, HIF1-α is stabilized during hypoxia, and its expression and transcriptional activity can be partially attenuated by concurrent imatinib treatment. Expression analysis demonstrates at the whole-transcriptome level that hypoxia and imatinib regulate distinct subsets of genes. Functionally, knockdown of HIF1-α abolished the enhanced clonogenicity during hypoxia. Taken together, our results suggest that in the hypoxic microenvironment, HIF1-α signaling supports LSC persistence independent of BCR-ABL1 kinase activity. Thus, targeting HIF1-α and its pathway components may be therapeutically important for the complete eradication of LSCs.

The RNA Pol II sub-complex hsRpb4/7 is required for viability of multiple human cell lines.

The evolutionarily conserved RNA Polymerase II Rpb4/7 sub-complex has been thoroughly studied in yeast and impacts gene expression at multiple levels including transcription, mRNA processing and decay. In addition Rpb4/7 exerts differential effects on gene expression in yeast and Rpb4 is not obligatory for yeast (S. cerevisiae) survival. Specialised roles for human (hs) Rpb4/7 have not been extensively described and we have probed this question by depleting hsRpb4/7 in established human cell lines using RNA interference. We find that Rpb4/7 protein levels are inter-dependent and accordingly, the functional effects of depleting either protein are co-incident. hsRpb4/7 exhibits gene-specific effects and cells initially remain viable upon hsRpb4/7 depletion. However prolonged hsRpb4/7 depletion is cytotoxic in the range of cell lines tested. Protracted cell death occurs by an unknown mechanism and in some cases is accompanied by a pronounced elongated cell morphology. In conclusion we provide evidence for a gene-specific role of hsRpb4/7 in human cell viability.

A common BIM deletion polymorphism mediates intrinsic resistance and inferior responses to tyrosine kinase inhibitors in cancer.

Tyrosine kinase inhibitors (TKIs) elicit high response rates among individuals with kinase-driven malignancies, including chronic myeloid leukemia (CML) and epidermal growth factor receptor-mutated non-small-cell lung cancer (EGFR NSCLC). However, the extent and duration of these responses are heterogeneous, suggesting the existence of genetic modifiers affecting an individual's response to TKIs. Using paired-end DNA sequencing, we discovered a common intronic deletion polymorphism in the gene encoding BCL2-like 11 (BIM). BIM is a pro-apoptotic member of the B-cell CLL/lymphoma 2 (BCL2) family of proteins, and its upregulation is required for TKIs to induce apoptosis in kinase-driven cancers. The polymorphism switched BIM splicing from exon 4 to exon 3, which resulted in expression of BIM isoforms lacking the pro-apoptotic BCL2-homology domain 3 (BH3). The polymorphism was sufficient to confer intrinsic TKI resistance in CML and EGFR NSCLC cell lines, but this resistance could be overcome with BH3-mimetic drugs. Notably, individuals with CML and EGFR NSCLC harboring the polymorphism experienced significantly inferior responses to TKIs than did individuals without the polymorphism (P = 0.02 for CML and P = 0.027 for EGFR NSCLC). Our results offer an explanation for the heterogeneity of TKI responses across individuals and suggest the possibility of personalizing therapy with BH3 mimetics to overcome BIM-polymorphism-associated TKI resistance.

A transcription assay for EWS oncoproteins in Xenopus oocytes.

Aberrant chromosomal fusion of the Ewing's sarcoma oncogene (EWS) to several different cellular partners produces the Ewing's family of oncoproteins (EWS-fusion-proteins, EFPs) and associated tumors (EFTs). EFPs are potent transcriptional activators, dependent on the N-terminal region of EWS (the EWS-activation-domain, EAD) and this function is thought to be central to EFT oncogenesis and maintenance. Thus EFPs are promising therapeutic targets, but detailed molecular studies will be pivotal for exploring this potential. Such studies have so far largely been restricted to intact mammalian cells while recent evidence has indicated that a mammalian cell-free transcription system may not support bona fide EAD function. Therefore, the lack of manipulatable assays for the EAD presents a significant barrier to progress. Using Xenopus laevis oocytes we describe a plasmid-based micro-injection assay that supports efficient, bona fide EAD transcriptional activity and hence provides a new vehicle for molecular dissection of the EAD.

In vitro activity of the EWS oncogene transcriptional activation domain.

Aberrant chromosomal fusion of the Ewings sarcoma oncogene (EWS) to several different cellular partners gives rise to the Ewing's family of oncogenic proteins [EWS fusion proteins (EFPs)] and associated tumors (EFTs). EFPs are potent transcriptional activators dependent on the N-terminal region of EWS [the EWS activation domain (EAD)], and this function is thought to be central to EFT oncogenesis and maintenance. Thus, EFPs are promising therapeutic targets, and detailed molecular studies of the EAD will be pivotal for exploring this potential. For many reasons, the molecular mechanism of EAD action is poorly understood and one major obstacle to progress is the lack of an in vitro transcription assay. Using well-characterized EAD-dependent activators and soluble nuclear extracts, we have attempted to recapitulate EAD transcriptional activity in vitro. We report that while the EAD activates transcription strongly in vitro, the effect of EAD mutations is strikingly different from that observed in vivo. Our results therefore suggest that crude soluble extracts do not support bona fide EAD activity in vitro, and we discuss our findings in relation to future assay development and potential mechanisms of EAD action.

Multiple aromatic side chains within a disordered structure are critical for transcription and transforming activity of EWS family oncoproteins.

Chromosomal translocations involving the N-terminal approximately 250 residues of the Ewings sarcoma (EWS) oncogene produce a group of EWS fusion proteins (EFPs) that cause several distinct human cancers. EFPs are potent transcriptional activators and interact with other proteins required for mRNA biogenesis, indicating that EFPs induce tumorigenesis by perturbing gene expression. Although EFPs were discovered more than a decade ago, molecular analysis has been greatly hindered by the repetitive EWS activation domain (EAD) structure, containing multiple degenerate hexapeptide repeats (consensus SYGQQS) with a conserved tyrosine residue. By exploiting total gene synthesis, we have been able to systematically mutagenize the EAD and determine the effect on transcriptional activation by EWS/ATF1 and cellular transformation by EWS/Fli1. In both assays, we find the following requirements for EAD function. First, multiple tyrosine residues are essential. Second, phenylalanine can effectively substitute for tyrosine, showing that an aromatic ring can confer EAD function in the absence of tyrosine phosphorylation. Third, there is little requirement for specific peptide sequences and, thus, overall sequence composition (and not the degenerate hexapeptide repeat) confers EAD activity. Consistent with the above findings, we also report that the EAD is intrinsically disordered. However, a sensitive computational predictor of natural protein disorder (PONDR VL3) identifies potential molecular recognition features that are tyrosine-dependent and that correlate well with EAD function. In summary we have uncovered several molecular features of the EAD that will impact future studies of the broader EFP family and molecular recognition by complex intrinsically disordered proteins.