Blastic Plasmacytoid Dendritic Cell Neoplasm Cells Research Articles

Transcriptional Landscape of CUT-Class Homeobox Genes in Blastic Plasmacytoid Dendritic Cell Neoplasm.

Homeobox genes encode developmental transcription factors regulating tissue-specific differentiation processes and drive cancerogenesis when deregulated. Dendritic cells (DCs) are myeloid immune cells occurring as two types, either conventional or plasmacytoid DCs. Recently, we showed that the expression of NKL-subclass homeobox gene VENTX is restricted to conventional DCs, regulating developmental genes. Here, we identified and investigated homeobox genes specifically expressed in plasmacytoid DCs (pDCs) and derived blastic plasmacytoid dendritic cell neoplasm (BPDCN). We analyzed gene expression data, performed RQ-PCR, protein analyses by Western blot and immuno-cytology, siRNA-mediated knockdown assays and subsequent RNA-sequencing and live-cell imaging. Screening of public gene expression data revealed restricted activity of the CUT-class homeobox gene CUX2 in pDCs. An extended analysis of this homeobox gene class in myelopoiesis showed that additional CUX2 activity was restricted to myeloid progenitors, while BPDCN patients aberrantly expressed ONECUT2, which remained silent in the complete myeloid compartment. ONECUT2 expressing BPDCN cell line CAL-1 served as a model to investigate its regulation and oncogenic activity. The ONECUT2 locus at 18q21 was duplicated and activated by IRF4, AUTS2 and TNF-signaling and repressed by BMP4-, TGFb- and IL13-signalling. Functional analyses of ONECUT2 revealed the inhibition of pDC differentiation and of CDKN1C and CASP1 expression, while SMAD3 and EPAS1 were activated. EPAS1 in turn enhanced survival under hypoxic conditions which thus may support dendritic tumor cells residing in hypoxic skin lesions. Collectively, we revealed physiological and aberrant activities of CUT-class homeobox genes in myelopoiesis including pDCs and in BPDCN, respectively. Our data may aid in the diagnosis of BPDCN patients and reveal novel therapeutic targets for this fatal malignancy.

Defining a Targetable Leukemia Intrinsic Dependency on Noncanonical PI3Kgamma Signaling

There are overlapping clinical and pathologic characteristics between poor prognosis subsets of acute myeloid leukemia (AML) and blastic plasmacytoid dendritic cell neoplasm (BPDCN), but common dependencies are ill-defined. We hypothesized that identifying novel shared targets and biomarkers could elucidate biology and nominate new therapies for both malignancies. We performed genome-wide CRISPR interference in BPDCN cells and identified 327 dependency genes, which were then analyzed informatically and functionally in AML, ALL, and BPDCN cells and patient-derived xenografts (PDXs). We defined a leukemia subset characterized by PIK3R5 activation and sensitivity to phosphoinositide 3-kinase gamma (PI3Kγ) inhibition via a noncanonical downstream pathway. The PI3Kγ complex includes an enzymatic subunit p110γ encoded by PIK3CG and a regulatory subunit p101 encoded by PIK3R5. Intriguingly, despite elevation of both p110γ and p101 proteins in this leukemia subset, only the PIK3R5 mRNA is overexpressed. To investigate this disconnection, we measured protein half-life and found that p110γ stability is positively regulated by PIK3R5/p101, associated with reduced p110γ ubiquitination. This suggested that increased expression of PIK3R5/p101 leads to posttranslational p110γ elevation by protecting it from ubiquitin proteasome-mediated degradation. To identify the upstream activator of PIK3R5, we analyzed RNA-seq data in >200 leukemia PDXs and found that AMLs with activated PIK3R5 are enriched for an innate immune response signature (IIRS). Supporting this model and indicating potential clinical relevance, analysis of TCGA AML patients revealed that the IIRS, which includes PIK3R5, was associated with M4/M5 monocytic AML and poor prognosis, independent of age, cytogenetics, or somatic mutations. Furthermore, in cells with low baseline expression, PIK3R5 was induced by the Toll-like receptor agonist resiquimod, showing that PIK3R5 is activated by inflammatory signaling and consistent with a monocytic/dendritic lineage association. By integrated analysis of ATAC-seq and ChIP-seq in PIK3R5 high vs low leukemias, as well as gene dependency scores, we identified the transcription factor PU.1 as responsible for PIK3R5 activation. Treatment with resiquimod enhanced the interaction between PU.1 and the PIK3R5 promoter, while depletion of PU.1 nullified the ability of resiquimod to activate PIK3R5. To elucidate molecular events downstream of p101/PI3Kγ, we studied consequences of pathway inactivation using PIK3R5 interference or the PI3Kγ-selective inhibitor eganelisib. Surprisingly, there was no effect of PI3Kγ inactivation on AKT phosphorylation or canonical PI3K-AKT-mTOR mRNA targets. Therefore, we performed phosphoproteomics and found that PAK1 S144, a phosphorylation site not previously associated with PI3K signaling, was suppressed by PIK3R5/PI3Kγ inhibition ( panel a). Selective inhibitors of PAK1, but not AKT, replicated the effect of eganelisib. Furthermore, introduction of constitutively active PAK1, but not inactive PAK1, eliminated sensitivity of leukemias to eganelisib, supporting the functional relationship between PAK1 phosphorylation and PI3Kγ dependency. PI3Kγ inhibitors are being tested in solid tumor trials to repolarize macrophages and potentiate immune checkpoint blockade, but their cancer cell-intrinsic role has not been explored. To assess PI3Kγ inhibition in vivo, we first performed intradermal xenografting assays using BPDCN derived from a patient's skin tumor. Eganelisib achieved comparable efficacy to cytarabine chemotherapy and the combination of eganelisib and cytarabine exerted significant synergy (combination index=0.63, p<0.0001). Next, we measured the effect of PI3Kγ inhibition on survival in disseminated PDX models. As predicted, single agent eganelisib significantly prolonged survival in AML, ALL, and BPDCN with high PIK3R5, while having no benefit in cases with low PIK3R5. To our surprise, the combination of eganelisib/cytarabine synergistically increased survival in all cases, regardless of baseline PIK3R5 ( panel b). We conclude that inflammatory signaling-activated PIK3R5 is a biomarker for sensitivity to PI3Kγ inhibition via an unappreciated PI3Kγ-PAK1 axis. Synergy with cytarabine unmasked eganelisib sensitivity in additional leukemias, expanding the therapeutic potential of targeting PI3Kγ.

Spatial Response to Pivekimab Sunirine (IMGN632) In Vivo in a BPDCN Model

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare, aggressive hematologic malignancy, which often manifests with skin lesions in addition to lymph node, blood, and bone marrow involvement. Standard of care treatments for BPDCN patients are intense chemotherapy or tagraxofusp (Elzonris®). Although many patients initially respond to standard-of-care therapies, most will eventually relapse. Overexpression of CD123 (IL-3Rα receptor) is a hallmark of BPDCN thus making this antigen an attractive target. Pivekimab sunirine is an antibody-drug-conjugate (ADC) comprised of a humanized anti-CD123 monoclonal antibody conjugated to a potent DNA-damaging agent, FGN849 via a cleavable linker. Pivekimab sunirine was granted orphan drug and Breakthrough Therapy designation and is currently being tested for the treatment of BPDCN patients as monotherapy and, as a triplet therapy in combination with azacitidine and venetoclax for the treatment of AML patients. Preclinically, pivekimab sunirine has demonstrated potent activity against BPDCN cells (Blood 2018 132:3956) and importantly, showed a favorable safety profile and promising efficacy in BPDCN patients (Hemasphere. 2022 Jun; 6 (Suppl): 1708-1709; ASH 2021, Abstract #1284). The goal of this study was to investigate the response to pivekimab sunirine in a pre-clinical model of BPDCN and to monitor spatial response in vivo. Pivekimab sunirine efficacy was evaluated in vitro on GEN2.2, a patient-derived BPDCN cell line, for 24h, 48h, or 72h at different concentrations. We observed a concentration and time-dependent efficacy at 48h or 72h of treatment with EC50s between 4pM and 12pM and between 1.3pM and 3pM respectively. Limited cytotoxicity was observed at the 24h time point. The efficacy of pivekimab sunirine was further confirmed in vivo in the GEN2.2 BPDCN model. Pivekimab sunirine treatment with either 0.024mg/kg or 0.08mg/kg doses at QWx3 (once weekly for 3 weeks) dosing regimen was able to restrain the tumor burden and significantly enhance survival compared to the vehicle group To assess spatial response to pivekimab sunirine, we utilized a bioluminescent (BLI) GEN2.2-luc model. Briefly, NSG mice were injected with 1x10 6 GEN2.2-luc cells followed by Fc receptor blockade on Day 2 for 24h. On Day 3, mice were treated with pivekimab sunirine (0.024mg/kg; QWx3) or vehicle intravenously. Organs were harvested on day 16 for the vehicle-treated group and day 30 for the pivekimab sunirine-treated group and tumor burden was assessed using luciferin. The vehicle group showed the presence of tumor cells in the bone marrow, spleen, and lungs. These results were confirmed by HES staining. Pivekimab sunirine treated mice showed no sign of tumor cells in any of the organs in BLI evaluation. HES staining confirmed the elimination of tumor cells in the lungs and the liver while few remaining tumor cells were observed in the spleen and the bone marrow. Overall, this work enabled us to validate the efficacy of pivekimab sunirine in a BPDCN model and to monitor its spatial effect. Data showed that pivekimab sunirine treatment significantly reduced tumor burden and increased survival compared to the vehicle group. Pivekimab sunirine treatment eradicated tumor cells in the lungs and the liver, while reducing the number of tumor cells in the spleen and bone marrow. Pivekimab sunirine is a potent ADC targeting CD123 and is highly efficacious against an aggressive BPDCN cell line model. This finding reinforces the importance of its use for the treatment of BPDCN patients.

Clonal Hematopoiesis Evolves into Dendritic Cell Neoplasm through the Acquisition of UV Mutations in the Skin

Background Expansion of blood cells derived from a single mutated blood stem cell - clonal hematopoiesis - often precedes leukemic transformation. However, the circumstances that promote or prevent transformation are poorly understood. Here, we investigate the evolution of clonal hematopoiesis into blastic plasmacytoid dendritic cell neoplasm (BPDCN), an aggressive blood cancer that often presents with malignant cells isolated to the skin (referred to as skin-only disease). We gain a better understanding of the influence of site-specific selective pressures and the role of TET2 mutations, which may inform interventions to prevent disease progression. Methods We analyzed bone marrow and skin from 13 BPDCN patients using targeted, exome, and whole-genome sequencing, with germline controls. We performed multimodal single-cell sequencing (10x 3' v3.1 single-cell RNA-seq with genotyping of expressed variants) on bone marrow from 6 healthy donors (20,411 cells), 5 skin-only BPDCN patients with malignant cells in the skin but not conventionally detectable in marrow (36,018 cells), and 6 BPDCN patients with overt bone marrow involvement (30,582 cells). For functional studies, we targeted Tet2 using CRISPR/Cas9 in mouse blood progenitor cells immortalized by an estrogen receptor hormone binding domain fused to HoxB8, which undergo synchronized differentiation to myeloid and dendritic cells upon estrogen withdrawal. We used CellRanger, Seurat, RandomForest machine learning, and the R tidyverse package collection for bioinformatics. Results Genetics allowed us to reconstruct the evolution of BPDCN across bone marrow and skin (representative patient, panel a). Invariably, we detected founder mutations in marrow, including TET2 inactivation in 9/13 patients (two mutations, likely bi-allelic, in 5/9). Single-cell sequencing of marrow from 5 skin-only patients showed that pre-malignant mutant stem cells contributed to multiple lineages, and that TET2 mutated clones were myeloerythroid-biased. We generated a gene expression signature to identify rare malignant BPDCN cells in marrow of skin-only patients. In 3/5 patients, we identified rare cells (0.03-0.19%) with the transcriptional profile of fully transformed cells, and concurrent single-cell genetic analysis suggested that these cells originated from the skin tumor (panel b). To assess whether disseminated skin tumor cells establish bone marrow disease, we quantified mutational signatures: skin tumors and malignant BPDCN marrow cells in 5/5 patients harbored UV-induced mutations, likely acquired in a single skin-resident plasmacytoid dendritic cell (pDC) during malignant transformation. Finally, we edited Tet2 in mouse myelo-dendritic progenitors and tested the response to UV irradiation in vitro. In addition to observing that Tet2 inactivation increases dendritic cell commitment, the more striking result was a survival advantage of Tet2-mutated cells in the presence of UV (n = 6, fold change 1.803, P = 0.0012). These results highlight an unexpected function of TET2 that may predispose mutated pDCs to survive UV-induced DNA damage in the skin, leading to disease progression. Conclusion The complex evolution of BPDCN traverses multiple anatomic sites prior to full transformation. This allows for accurate sequencing of oncogenic events. Disease evolution frequently starts with acquisition of a leukemia-associated mutation in a multipotent blood stem cell, leading to clonal expansion. In the most frequently observed scenario, bi-allelic TET2 inactivation promotes myeloerythroid bias, increases dendritic cell commitment, and confers resistance to UV-mediated cell death. Subsequent evolution of skin-resident mutant pDCs leads to a high burden of UV-induced mutations and ultimate transformation. Dissemination of malignant cells, which can be detected by integrating single-cell gene expression and mutation profiles, initiates "retrograde" bone marrow disease. Our discovery that multiple anatomic sites - and local selective pressures - play a role in disease progression provides a template to study multi-site evolution of clonal hematopoiesis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Blastic plasmacytoid dendritic cell neoplasm (BPDCN): A promising future in the era of targeted therapeutics.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematologic malignancy arising from precursor dendritic cells. BPDCN cells characteristically express several markers on their cell surfaces including CD123, CD4, and CD56. Because of its rarity and challenging clinical presentation, there was no standard of care in managing BPDCN for decades and its prognosis overall was poor. However, as understanding of this rare neoplasm has increased, so have treatment options. The conventional cytotoxic chemotherapy regimens once used in the treatment of BPDCN were modest in their impact on disease relapse until paired with hematopoietic stem cell transplant. Although recent data suggest that there still remains a role for chemotherapeutic agents, targeted modalities have expanded the overall BPDCN treatment landscape. The CD123-targeted agent, tagraxofusp, was the first Food and Drug Administration-approved monotherapy in the treatment of BPDCN. Since its inception, several CD123-targeted and other cell-surface agents have been investigated, with many agents still in the preclinical stages. Although relapsed/refractory disease and central nervous system disease both remain formidable areas of research, there are several promising therapeutic approaches that could have a significant impact on the trajectory of treatment. This review will provide detailed insight on the novel drugs currently in use and those being explored in the management of BPDCN.

Targeting CD123 in blastic plasmacytoid dendritic cell neoplasm using allogeneic anti-CD123 CAR T cells

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematologic malignancy with poor outcomes with conventional therapy. Nearly 100% of BPDCNs overexpress interleukin 3 receptor subunit alpha (CD123). Given that CD123 is differentially expressed on the surface of BPDCN cells, it has emerged as an attractive therapeutic target. UCART123 is an investigational product consisting of allogeneic T cells expressing an anti-CD123 chimeric antigen receptor (CAR), edited with TALEN® nucleases. In this study, we examine the antitumor activity of UCART123 in preclinical models of BPDCN. We report that UCART123 have selective antitumor activity against CD123-positive primary BPDCN samples (while sparing normal hematopoietic progenitor cells) in the in vitro cytotoxicity and T cell degranulation assays; supported by the increased secretion of IFNγ by UCART123 cells when cultured in the presence of BPDCN cells. UCART123 eradicate BPDCN and result in long-term disease-free survival in a subset of primary patient-derived BPDCN xenograft mouse models. One potential challenge of CD123 targeting therapies is the loss of CD123 antigen through diverse genetic mechanisms, an event observed in one of three BPDCN PDX studied. In summary, these results provide a preclinical proof-of-principle that allogeneic UCART123 cells have potent anti-BPDCN activity.

Single-Cell Multiomics Reveals Clonal T-Cell Expansions and Exhaustion in Blastic Plasmacytoid Dendritic Cell Neoplasm.

The immune system represents a major barrier to cancer progression, driving the evolution of immunoregulatory interactions between malignant cells and T-cells in the tumor environment. Blastic plasmacytoid dendritic cell neoplasm (BPDCN), a rare acute leukemia with plasmacytoid dendritic cell (pDC) differentiation, provides a unique opportunity to study these interactions. pDCs are key producers of interferon alpha (IFNA) that play an important role in T-cell activation at the interface between the innate and adaptive immune system. To assess how uncontrolled proliferation of malignant BPDCN cells affects the tumor environment, we catalog immune cell heterogeneity in the bone marrow (BM) of five healthy controls and five BPDCN patients by analyzing 52,803 single-cell transcriptomes, including 18,779 T-cells. We test computational techniques for robust cell type classification and find that T-cells in BPDCN patients consistently upregulate interferon alpha (IFNA) response and downregulate tumor necrosis factor alpha (TNFA) pathways. Integrating transcriptional data with T-cell receptor sequencing via shared barcodes reveals significant T-cell exhaustion in BPDCN that is positively correlated with T-cell clonotype expansion. By highlighting new mechanisms of T-cell exhaustion and immune evasion in BPDCN, our results demonstrate the value of single-cell multiomics to understand immune cell interactions in the tumor environment.

Evaluating tagraxofusp for the treatment of blastic plasmacytoid dendritic cell neoplasm (BPDCN)

ABSTRACT Introduction Unique among hematologic malignancies, blastic plasmacytoid dendritic cell neoplasm (BPDCN) affects multiple compartments including bone marrow, hematologic, lymphatic, dermatologic, and central nervous systems (CNS). Treating BPDCN is challenging, historically, as patients display refractoriness to chemotherapy and absence of long-term remissions in many cases not treated with hematopoietic stem cell transplantation. Discovering the prevalent overexpression of surface receptor CD123 (IL3-Rα) on BPDCN cells led to development of tagraxofusp, a novel anti-CD123 agent for patients with BPDCN. Areas covered Herein, the authors discuss the preclinical development and phase I/II clinical studies, which led to the approval of tagraxofusp. They discuss the current treatment landscape of BPDCN with tagraxofusp alone or combined with chemotherapy and highlight several ongoing clinical trials involving combinations of tagraxofusp with novel targeted therapeutics for BPDCN. Expert opinion Tagraxofusp has significantly improved the treatment landscape for patients with newly diagnosed BPDCN and has led to investigative efforts and augmentation of various strategies of targeting CD123, such as antibody-drug conjugates and anti-CD123 chimeric antigen receptor T-cells. However, relapsed/refractory disease and CNS-involvement of BPDCN remain therapeutic challenges. The authors recommend that healthcare providers consider multiple-agent clinical trial approaches and adequate CNS-prophylaxis for all patients with BPDCN.

Loss of METTL3 Attenuates BPDCN Response to PRMT5 Inhibition Via Interferon Signaling

Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN) is an aggressive hematologic cancer that stems from plasmacytoid dendritic cells (pDCs) and predisposes to leukemic transformation. Other than Tagraxofusp-erzs, a recently approved targeted therapy, treatment options for BPDCN remain inadequate. Recently, it was reported that BPDCN patient samples were enriched for the PRMT5 gene signature, suggesting a role for PRMT5 in BPDCN. Pursuing the hypothesis that targeting PRMT5 represents a potential therapeutic option in BPDCN, we first clarified the implications of inhibiting PRMT5‘s activity using a small molecule inhibitor - GSK3326595 (GSK595). Our previous findings revealed that perturbations to PRMT5's activity dysregulated the splicing landscape and mitigated tumor growth. Additionally, we identified RNA methylation as a process affected by PRMT5 inhibition.Presently, to follow up on RNA methylation's role in BPDCN we focused on METTL3 - a key RNA modifying enzyme. CRISPR-Cas9 mediated knockout (KO) of METTL3 in CAL-1 (BPDCN patient-derived cell line) was found to affect CAL-1 cell growth, similar to PRMT5 inhibition. To ascertain if PRMT5 inhibition in these KO cells exacerbates the effects on cell growth, we treated METTL3 KO cells with GSK595. Unexpectedly, GSK595-treated cells displayed reduced sensitivity to PRMT5 inhibition. This was supported by the lack of decreased cell growth and diminished apoptosis activation, despite dose-dependent decrease in SDMA expression, signifying that GSK595 still perturbs PRMT5 activity in these cells. To uncover the mechanism underlying this unique finding, we performed transcriptomic analyses of untreated and treated METTL3 KO cells. RNA-seq results showed that Type 1 interferon (IFN) signaling was highly upregulated in KO cells upon GSK595 treatment. Volcano plot analysis then identified Interferon Induced Protein with Tetratricopeptide Repeats 1 (IFIT1), an Interferon-stimulated gene (ISG) to be highly upregulated post treatment and this finding was validated in vitro as well. This now highlights the possibility that increased Type 1 IFN signaling might confer resistance against PRMT5 inhibition in BPDCN cells with low/no METTL3 expression. To gain further insight into this observation, we assessed the expression of Interferon Regulatory Factor 7 (IRF7), the transcription factor regulating expression of ISGs like IFIT1. Upon PRMT5 inhibition in WT CAL-1 cells, IRF7 displayed dysregulated splicing (intron retention), similar to METTL3. In METTL3 KO cells, like IFIT1, the expression of IRF7 increased significantly post GSK595 treatment. This implies that the IRF7-IFIT1 axis is controlled by the expression status of METTL3 and this in turn affects response to PRMT5 inhibition. To verify this functionally, we added TLR7 agonists to CAL-1 cells to induce Type 1 IFN signaling and concurrently treated these cells with GSK595. Comparable to METTL3 KO cells, these TLR7-induced cells showed rescue in cell growth post treatment and had very weak apoptosis activation.Overall, the findings from this study shows that the status of METTL3 affects BPDCN cells’ response to pharmacologic modulation of PRMT5's activity by regulating IFN signaling via the IRF7-IFIT1 axis. Thus, implying that stratification based on METTL3 status may help select BPDCN patients who might respond optimally to PRMT5-targted therapies. DisclosuresNo relevant conflicts of interest to declare.

A Study of IMGN632, a Novel CD123-Targeting Antibody-Drug Conjugate, for Patients with Frontline and Relapsed/Refractory Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN)

A Study of IMGN632, a Novel CD123-Targeting Antibody-Drug Conjugate, for Patients with Frontline and Relapsed/Refractory Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN)

Newly-Discovered Neural Features Expand the Pathobiological Knowledge of Blastic Plasmacytoid Dendritic Cell Neoplasm.

Simple SummaryFor the first time, neuronal features are described in blastic plasmacytoid dendritic cell neoplasm (BPDCN) by a complex array of molecular techniques, including microRNA and gene expression profiling, RNA and Chromatin immunoprecipitation sequencing, and immunohistochemistry. The discovery of unexpected neural features in BPDCN may change our vision of this disease, leading to the designing of a new BPDCN cell model and to re-thinking the relations occurring between BPDCN and nervous system. The observed findings contribute to explaining the extreme tumor aggressiveness and also to propose novel therapeutic targets. In view of this, the identification, in this work of new potential neural metastatic inducers might open the way to therapeutic approaches for BPDCN patients based on the use of anti-neurogenic agents.Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and highly aggressive hematologic malignancy originating from plasmacytoid dendritic cells (pDCs). The microRNA expression profile of BPDCN was compared to that of normal pDCs and the impact of miRNA dysregulation on the BPDCN transcriptional program was assessed. MiRNA and gene expression profiling data were integrated to obtain the BPDCN miRNA-regulatory network. The biological process mainly dysregulated by this network was predicted to be neurogenesis, a phenomenon raising growing interest in solid tumors. Neurogenesis was explored in BPDCN by querying different molecular sources (RNA sequencing, Chromatin immunoprecipitation-sequencing, and immunohistochemistry). It was shown that BPDCN cells upregulated neural mitogen genes possibly critical for tumor dissemination, expressed neuronal progenitor markers involved in cell migration, exchanged acetylcholine neurotransmitter, and overexpressed multiple neural receptors that may stimulate tumor proliferation, migration and cross-talk with the nervous system. Most neural genes upregulated in BPDCN are currently investigated as therapeutic targets.

Preclinical Studies Of SL-401, a Targeted Therapy Directed To The Interleukin-3 Receptor (IL3-R), In Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN): Potent Activity In BPDCN Cell Lines, Primary Tumor, and In An In Vivo Model

Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN), an aggressive malignancy derived from plasmacytoid dendritic cells (PDCs), typically presents as cutaneous lesions, can also involve lymph nodes and spleen, and invariably terminates in an acute leukemic phase. Its diagnosis is based on this clinical presentation, as well as the unique pathological and immunophenotypic features of a cutaneous PDC neoplasm, with high expression of CD123 (interleukin-3 receptor [IL-3R]), CD304, and/or CD303 (Garnache-Ottou F et al., 2009); CD56 and CD4 are also commonly expressed. There is no accepted standard of care to treat BPDCN and therapeutic strategies have never been prospectively evaluated. Treatment approaches include: symptomatic therapy, intensive multi-agent chemotherapy, and allogenic hematopoietic cell transplantation. Although chemotherapy-naïve patients often respond to chemotherapy, most subjects experience recurrent disease shortly thereafter; median overall survival ranges from 9 to 13 months. Since BPDCN cells express high levels of IL-3R, the antitumor effects of SL-401, a novel targeted therapy directed to IL-3R, are being evaluated in preclinical and clinical studies. SL-401, which consists of IL-3 fused to a truncated diphtheria toxin payload, potently inhibits protein synthesis following IL-3R-mediated binding and internalization. Since normal hematopoietic cells express negligible levels of IL-3R, myelosuppression is negligible. To better understand the antitumor effects of SL-401 against BPDCN, the cytotoxicity of SL-401 was assessed in BPDCN patient-derived cell lines (CAL-1 and GEN2.2) and primary BPDCN cells isolated directly from patients (n = 12). Cytotoxicity was measured using flow cytometry (AV/7AAD staining) and an MTT assay after SL-401 treatment for 18 or 48 hours, respectively, as a single agent or compared with a range of cytotoxic agents. We confirmed the robust single agent activity of SL-401 against BPDCN cell lines (Angelot-Delettre F et al., ASH 2011) and found that treatment with SL-401 at concentrations substantially lower than peak concentrations achieved in patients induced a percent decrease in viability of BPDCN cell lines of 92% (62 ± 7% to 5 ± 2%) (n = 9 experiments) and primary BPDCN cells of 80% (50 ± 5% to 10 ± 1%) (n = 12 experiments) (Fig. A and B). Moreover, SL-401 was more effective than 7 out of 8 chemotherapeutic agents tested (Fig. C). SL-401 was also evaluated in a NOD-SCID IL2RγcKO (NSG) BPDCN mouse model. After 2 Gy irradiation, mice were injected with GEN 2.2 cells (1 x 106, IV) and then treated with SL-401 at clinically relevant doses or PBS (IP daily for 5 days) initiated on day 7 post-implantation. The median overall survival (OS) was significantly improved in mice receiving a single cycle of SL-401 compared to untreated controls (53 d vs. 15 d, respectively) (Figs. D and E). These findings provide strong in vitro and in vivo evidence that BPDCN cells are highly sensitive to SL-401. Since BPDCN patients express high levels of IL-3R (Garnache Ottou et al, 2009), these results also indicate that SL-401 may provide a clinical benefit for BPDCN patients. A Phase 2 multi-cycle trial in this orphan indication is currently being planned in North America and Europe. [Display omitted] (A) BPDCN cell lines CAL-1 and GEN 2.2 (n= 9) and (B) primary cells (12 patients) are sensitive to SL-401 (2.7 fM) treatment (18 h) in vitro. **p<0.001. (C) BPDCN cells were cultured alone or with SL-401 (2.7 fM) or ERW (erwinase, 10 UI/ml), KID (kidrolase, 10UI/ml), MTX (methotrexate, 4.5µg/ml), CYC (cyclophosphamide, 100 µmM), CYT (cytarabine, 80 µg/ml), DEX (dexamethasone, 250 µg/ml), VIN (vincristine, 20 ng/ml), or IDA (idarubicin, 0.078 µg/ml). The viability was evaluated by cytometry using AV/7AAD staining after 18 h of culture. Results (mean + SEM) of 4 experiments.*p<0.05. (D) Survival rate of 2 Gy irradiated NSG mice injected IV with 1 x 106 GEN 2.2 cells on day 0. SL-401 treatment IP (2 µg) daily for 5 days (pink area) was initiated on day 7. The solid and dotted lines show saline-treated controls (n = 3) and SL-401 treated animals (SL-401, n=4), respectively. Results from one representative experiment out of 3. (E) GEN 2.2 cells detected in a node 2 weeks post-injection. Cells were stained with standard MGG solution and show a blastic morphology. Disclosures:Frankel:Stemline Therapeutics: Patents & Royalties, Research Funding. Brooks:Stemline Therapeutics: Employment, Equity Ownership. Rowinsky:Stemline Therapeutics: Employment, Equity Ownership.

Pre-Clinical Studies of Allogeneic Anti-CD123 CAR T-Cells for the Therapy of Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN)

Pre-Clinical Studies of Allogeneic Anti-CD123 CAR T-Cells for the Therapy of Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN)

Male-Biased Spliceosome Mutations in Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN) Impair pDC Activation and Apoptosis

Male-Biased Spliceosome Mutations in Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN) Impair pDC Activation and Apoptosis

PRMT5 Inhibition Affects RNA Methylation and Is a Potential Therapeutic Target in BPDCN

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and clinically aggressive hematologic cancer that arises from malignant transformation of plasmacytoid dendritic cells (pDCs) and is predisposed to leukemic transformation. Apart from the recently approved therapy, Tagraxofusp-erzs, treatment options for BPDCN remain limited. A recurrent feature of BPDCN is dysregulated MYC expression, which is associated with dependence on Protein arginine methyltransferase 5 (PRMT5). PRMT5 catalyses symmetric dimethylation of arginine residues post translationally and is often overexpressed in hematological malignancies. It was recently reported that BPDCN patient samples were enriched for a PRMT5 gene signature. However, the role of elevated PRMT5 in BPDCN has not been studied. Moreover, how pharmacological inhibition of PRMT5 affects BPDCN remains unexplored. Here, we pursue the hypothesis that PRMT5 or PRMT5-regulated factors represent a potential therapeutic opportunity in BPDCN, a hematologic malignancy of unmet clinical need. We report that elevated PRMT5 activity is critical for BPDCN. Compared to pDCs from healthy individuals, BPDCN cells expressed much higher levels of MYC and PRMT5. Functionally, in vitro growth of BPDCN cells treated with the PRMT5 inhibitor GSK3326595 (GSK595) was impaired in a dose-dependent manner, with net negative cell growth observed 6 days post-treatment. Similarly, bi-daily oral administration of GSK595 to CAL-1 xenografts significantly mitigated the growth of tumors, without any observed impact on body weight. In addition, the sensitivity of BPDCN cells to GSK595 was comparable to MOLM-13, which has been reported to be highly sensitive to PRMT5 inhibition. Correspondingly, this growth inhibition was associated with the induction of apoptosis. Thus, confirming the requirement for elevated PRMT5 levels in BPDCN. We then transcriptionally profiled GSK595-treated BPDCN cells and identified differential splicing events (DSEs) across several splicing classes; of which intron retention (and to a lesser extent, exon skipping) events were substantially elevated. Interestingly, these upregulated intron retention events were over-represented for processes associated with RNA modification. Specifically, we found that splicing of several essential genes involved in RNA modification were dysregulated. Among these genes was Methyltransferase-like protein 3 (METTL3), an RNA methyltransferase writer which mediates m6A modification on mRNAs. In agreement, we show that PRMT5 inhibition decreased the expression of METTL3 along with its downstream targets - MYC &amp; TAZ. We also show that the levels of m6A modification in GSK595-treated BPDCN cells decreased in a dose-dependent manner; thus, highlighting the reduced functional activity of METTL3 following PRMT5 inhibition. Taken together, our data implicates METTL3 and possibly other essential RNA modification genes as key downstream effectors of PRMT5 in BPDCN. Collectively, our study highlights a novel link between PRMT5 and RNA modification in BPDCN. In addition, we show that elevated PRMT5 is important in BPDCN and its inhibition mitigates BPDCN progression. Therefore, PRMT5 inhibition is a potential therapeutic strategy for BPDCN. Disclosures No relevant conflicts of interest to declare.

Tagraxofusp and anti-CD123 in blastic plasmacytoid dendritic cell neoplasm: a new hope.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematological malignancy, characterized by poor prognosis if treated with conventional therapy. Allogenic hematologic stem cell transplant can improve survival and can be curative, but it is available in a small percentage of patients given that the median age at diagnosis is 70 years. In this scenario it is assumed that only the development of precision medicine-driven therapy will change BPDCN patient prognosis. CD123 (the α-subunit of interleukin (IL)-3 receptor) is over-expressed on BPDCN cells surface and seems to be the ideal marker to develop antibody-based therapies. Tagraxofusp (Elzonris®), a recombinant immunotoxin consisting of human interleukin-3 fused to a truncated diphtheria toxin, has been approved by FDA in December 2018 for the treatment of BPDCN in adult and pediatric patients. tagraxofusp has shown promising clinical activity, with a high overall response rate and quite manageable safety profile even in elderly patients. It seems to improve overall survival too, but comparative trials are necessary to confirm this. Adverse events are commonly reported and the most important are transaminitis, thrombocytopenia and capillary leak syndrome (CLS). Therefore, to prevent the onset of severe CLS is recommended to reserve tagraxofusp for patients with preserved hepatic and cardiac functions, and to strictly observe serum albumin level. Further studies are required to resolve many several unanswered questions about tagraxofusp. In this review, we will resume and discuss pharmacological characteristic of tagraxofusp, results of clinical trials leading to its approval by FDA in 2018 and future perspectives about its use in BPDCN and other hematological malignancies.

Immunophenotypic characterization of reactive and neoplastic plasmacytoid dendritic cells permits establishment of a 10-color flow cytometric panel for initial workup and residual disease evaluation of blastic plasmacytoid dendritic cell neoplasm.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematopoietic neoplasm whose immunophenotype remains incompletely characterized, particularly with regards to its distinction from reactive plasmacytoid dendritic cells (PDC). This limitation complicates detection of low-level involvement by BPDCN as well as minimal residual disease (MRD) assessment following therapy. We conducted the current study to characterize the immunophenotype of BPDCN in a cohort of 39 patients, and compared it to that of reactive PDC. We found that, in addition to CD56 expression (97%), BPDCN showed a number of aberrancies, including decreased/negative CD38 (82%), positive CD7 (64%), negative CD2 (81%), negative CD303 (56%), increased HLA-DR (69%) and decreased CD123 (78%) expression. Although BPDCN cells were characterized by CD56 expression, reactive PDC consistently included a CD56+ subset, ranging from 1.3%-20% (median 4.5%) of all PDC, challenging the detection of MRD. These CD56+ reactive PDC were, however, consistently positive for CD2 and CD303, brightly positive for CD38, and negative for CD7, distinctively different from BPDCN. Based on these findings, we set up a ten-color flow cytometry assay for BPDCN and validated it to a sensitivity of 0.01%. This panel was prospectively tested in 19 bone marrow samples from seven patients with BPDCN, and it effectively distinguished BPDCN cells from background reactive PDC in all cases. In summary, by understanding the immunophenotype of reactive and neoplastic PDC, BPDCN can be effectively detected by flow cytometry to a very low level using a panel of markers in addition to CD56. Such an assay could be used for initial bone marrow workup as well as MRD detection after therapy.

Abstract PR14: CD123CAR displays clinical activity in relapsed/refractory (r/r) acute myeloid leukemia (AML) and blastic plasmacytoid dendritic cell neoplasm (BPDCN): Safety and efficacy results from a phase 1 study

Abstract Introduction: The current treatment of R/R AML and BPDCN is associated with poor outcome. CD123 is overexpressed on AML blasts, leukemic stem cells, and BPDCN cells compared to normal hematopoietic stem cells. This differential expression feature makes CD123 an attractive target for cellular immunotherapy. Here we report results of an ongoing single-center, first-in-human phase 1 dose-escalation study (NCT02159495) evaluating CD123CAR T cells in treating patients with R/R AML (cohort 1) and BPDCN (cohort 2). This study is designed to determine the safety and antileukemic activity of CD123CAR T cells. Methods: Vectors containing the CD123CAR composed of a single-chain variable fragment, an optimized IgG4 linker, a CD28 co-stimulatory domain, and a CD3 zeta domain are used to engineer donor-derived or autologous T cells via lentiviral transduction. Prior to T-cell infusion, all patients undergo lymphodepleting regimen with the majority receiving fludarabine 25-30 mg/m2 and cyclophosphamide 300-500 mg/m2 daily for 3 days. Patients can receive a second infusion if they continue to meet eligibility criteria. To date, 18 patients have been enrolled and 9 treated (7 AML, 2 BPDCN). Results: All 7 patients in the AML cohort had at least one prior allogeneic stem cell transplant and median of 4 (range: 4-10) prior lines of therapy. Of the 2 patients treated at the dose level (DL) 0 (50M CAR+ T), one achieved a morphologic leukemic-free state (MLFS), which lasted 70 days. She received a second infusion 3 months later with blast reduction from 77.9% to 0.9% (flow cytometry) after 35 days. Of the 5 patients on the DL1 (200M CAR+ T), 1 achieved complete remission (CR) with incomplete count recovery at day 28, and 1 MLFS that improved to CR at day 84. The remaining 3 patients had stable disease. All toxicities were reversible and manageable. No patient developed grade 3 or above cytokine release syndrome or neurotoxicity. There were no treatment-related dose-limiting toxicities and no treatment-related cytopenias longer than 12 weeks. One patient with prior cutaneous GVHD developed grade 1 rash 5 weeks after CAR T infusion, which resolved after clinical management. Peak of T-cell expansion occurred within the first 14 days. We did not observe any CD123-loss leukemic variants. In the BPDCN cohort, 2 patients treated at DL0 (100M CAR+ T) tolerated the treatment well with no grade 3 or above treatment-related toxicities. One patient achieved CR with no evidence of disease in the bone marrow and skin at day 28. Conclusions: In this first-in-human clinical trial of CD123CAR T cell therapy, we have demonstrated the feasibility, safety, and promising antileukemic activity of targeting CD123 in patients with AML or BPDCN. Based on the results we have observed and our collaborative work with Mustang Bio at optimizing the manufacturing platform, we will be leading a multicenter phase 1/2 clinical trial to determine the activity of the cells in achieving remission in patients with AML, BPDCN, and myelodysplastic syndrome. This abstract is also being presented as Poster B22. Citation Format: Lihua E. Budde, Joo Song, Marissa Del Real, Young Kim, Candida Toribio, Brent Wood, Jamie Wagner, Emanuela Marcucci, Anthony Stein, Guido Marcucci, Christine E. Brown, Stephen J. Forman. CD123CAR displays clinical activity in relapsed/refractory (r/r) acute myeloid leukemia (AML) and blastic plasmacytoid dendritic cell neoplasm (BPDCN): Safety and efficacy results from a phase 1 study [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr PR14.

CD28/4-1BB CD123 CAR T cells in blastic plasmacytoid dendritic cell neoplasm.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is associated with a remarkably poor prognosis and with no treatment consensus. The identification of relevant therapeutic targets is challenging. Here, we investigated the immune functions, antileukemia efficacy and safety of CD28/4-1BB CAR T cells targeting CD123 the interleukin (IL)-3 receptor alpha chain which is overexpressed on BPDCN. We demonstrated that both retroviral and lentiviral engineering CD28/4-1BB CD123 CAR T cells exhibit effector functions against BPDCN cells through CD123 antigen recognition and that they efficiently kill BPDCN cell lines and BPDCN-derived PDX cells. In vivo, CD28/4-1BB CD123 CAR T-cell therapy displayed strong efficacy by promoting a decrease of BPDCN blast burden. Furthermore we showed that T cells from BPDCN patient transduced with CD28/4-1BB CD123 CAR successfully eliminate autologous BPDCN blasts in vitro. Finally, we demonstrated in humanized mouse models that these effector CAR T cells exert low or no cytotoxicity against various subsets of normal cells with low CD123 expression, indicating a potentially low on-target/off-tumor toxicity effect. Collectively, our data support the further evaluation for clinical assessment of CD28/4-1BB CD123 CAR T cells in BPDCN neoplasm.

Efficacy Proof of Concept for Allogeneic CD123 Targeting CAR T-Cells Against Primary Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN): Efficient Control of Tumor Progression in PDX Model and Potential Loss of CD123 Expression in Relapsed Disease

Efficacy Proof of Concept for Allogeneic CD123 Targeting CAR T-Cells Against Primary Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN): Efficient Control of Tumor Progression in PDX Model and Potential Loss of CD123 Expression in Relapsed Disease