Human Hematologic Malignancies Research Articles

Applications and opportunities for immune cell CAR engineering in comparative oncology.

Chimeric antigen receptor (CAR) T adoptive cell therapy has transformed the treatment of human hematologic malignancies. However, its application for the treatment of solid tumors remains challenging. An exciting avenue for advancing this field lies in the use of pet dogs, in which cancers that recapitulate the biology, immunological features, and clinical course of human malignancies arise spontaneously. Moreover, their large size, outbred genetic background, shared environment with humans, and immunocompetency make dogs ideal for investigating and optimizing CAR therapies before human trials. Here, we will outline how challenges in early clinical trials in canine lymphoma patients, including issues related to autologous CAR-T cell manufacturing, limited CAR-T cell persistence, and tumor antigen escape, mirrored challenges observed in human CAR-T trials. We will then highlight emerging adoptive cell therapy strategies currently under investigation in dogs with hematological and solid cancers, that will provide crucial safety and efficacy data on novel CAR-T regimens that can be used to support clinical trials. By drawing from ongoing studies, we will illustrate how canine patients with spontaneous cancer may serve as compelling screening platforms to establish innovative CAR-therapy approaches and identify predictive biomarkers of response, with a specific emphasis on solid tumors. With increased funding for canine immunotherapy studies, multi-institutional investigations are poised to generate highly impactful clinical data that should translate into more effective human trials, ultimately benefiting both human and canine cancer patients.

Abstract CT116: Preclinical antitumor activity and first-in-human phase I study of NB004/GDC-0570, a novel pan-PIM kinase inhibitor, in patients with advanced solid tumors

Abstract PIM proto-oncogenes encode a family of three serine/threonine kinases (PIM1, PIM2, PIM3), which are frequently overexpressed in human hematological malignancies and solid cancers. PIM kinases are promising cancer therapeutic targets for pharmacological inhibition. NB004 (aka GDC-0570) is a novel, potent, selective, orally available small molecule pan-PIM inhibitor, which exhibits antiproliferative activity as a single agent in a variety of preclinical models and can augment the activity of standard of care or targeted therapies, both in vitro and in vivo. In multiple patient-derived xenograft (PDX) models of human solid tumors, NB004 demonstrates both single agent and combination activity with agents targeting the RTK/RAS/MAPK pathways, including synergistic effects with KRAS inhibitors (KRASis). Remarkably, in NSCLC PDX models with acquired resistance to KRASis, strong combination efficacy was observed between NB004 and sotorasib, resulting in complete tumor regression. Based on the promising preclinical data, NB004 is currently being assessed in an open-label, first-in-human phase I clinical trial as monotherapy or combination therapy in patients with advanced solid tumors. The primary objective is to characterize the safety and tolerability and to recommend the phase 2 dose (RP2D). Secondary objectives include PK characterization and preliminary antitumor activity evaluation. In the phase I monotherapy dose escalation part, NB004 was administered daily in 5 dose levels, ranging from 75mg to 600mg, following a standard 3+3 design. As of December 22, 2023, a total of 18 patients were treated. Of the 13 patients (72.2%) who reported any treatment-related adverse events (TRAEs), 12 (66.7%) had Grade 1 or 2 TRAEs, 1 patient (5.6%) experienced Grade 3 neutrophil count decrease and white blood cell count decrease. No dose-limiting toxicity (DLT) or treatment-related serious adverse event (SAE) was reported, and PK exposure is in general dose proportional. These results warrant further evaluation of this drug in combination with other therapeutic agents. The combination part of the trial is currently open for enrollment (NCT05036291). Citation Format: Siqing Fu, Rachel E. Sanborn, Minxia Cai, Lijia Zhang, Yanhua Xu, Kui Lin. Preclinical antitumor activity and first-in-human phase I study of NB004/GDC-0570, a novel pan-PIM kinase inhibitor, in patients with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT116.

Targeting STAT3 Enzyme for Cancer Treatment.

A category of cytoplasmic transcription factors called STATs mediates intracellular signaling, which is frequently generated at receptors on cell surfaces and subsequently sent to the nucleus. STAT3 is a member of a responsible for a variety of human tumor forms, including lymphomas, hematological malignancies, leukemias, multiple myeloma and several solid tumor types. Numerous investigations have demonstrated constitutive STAT3 activation lead to cancer development such as breast, head and neck, lung, colorectal, ovarian, gastric, hepatocellular, and prostate cancers. It's possible to get a hold of the book here. Tumor cells undergo apoptosis when STAT3 activation is suppressed. This review highlights the STAT3 activation and inhibition which can be used for further studies.

A New Synthesized Dicarboxylated Oxy-Heparin Efficiently Attenuates Tumor Growth and Metastasis.

Heparanase (Hpa1) is expressed by tumor cells and cells of the tumor microenvironment and functions to remodel the extracellular matrix (ECM) and regulate the bioavailability of ECM-bound factors that support tumor growth. Heparanase expression is upregulated in human carcinomas, sarcomas, and hematological malignancies, correlating with increased tumor metastasis, vascular density, and shorter postoperative survival of cancer patients, and encouraging the development of heparanase inhibitors as anti-cancer drugs. Among these are heparin/HS mimetics, the only heparanase-inhibiting compounds that are being evaluated in clinical trials. We have synthesized dicarboxylated oxy-heparins (DCoxHs) containing three carboxylate groups per split residue (DC-Hep). The resulting lead compound (termed XII) was upscaled, characterized, and examined for its effectiveness in tumor models. Potent anti-tumorigenic effects were obtained in models of pancreatic carcinoma, breast cancer, mesothelioma, and myeloma, yielding tumor growth inhibition (TGI) values ranging from 21 to 70% and extending the survival time of the mice. Of particular significance was the inhibition of spontaneous metastasis in an orthotopic model of breast carcinoma following resection of the primary tumor. It appears that apart from inhibition of heparanase enzymatic activity, compound XII reduces the levels of heparanase protein and inhibits its cellular uptake and activation. Heparanase-dependent and -independent effects of XII are being investigated. Collectively, our pre-clinical studies with compound XII strongly justify its examination in cancer patients.

Effect of proteasome inhibitors on canine lymphoma cell response to CHOP chemotherapy in vitro.

The standard treatment for canine lymphoma is the CHOP chemotherapy regimen. Proteasome inhibitors have been employed with CHOP for the treatment of human haematological malignancies but remain to be fully explored in canine lymphoma. We identified an association between poor response to CHOP chemotherapy and high mRNA expression levels of proteasomal subunits in a cohort of 15 canine lymphoma patients, and sought to determine the effect of proteasome inhibitors on the viability of a canine B-cell lymphoma cell line (CLBL-1). The aim of this study was to investigate whether proteasome inhibitors sensitize these cells to the CHOP agents doxorubicin, vincristine and cyclophosphamide (as 4-hydroxycyclophosphamide/4-HC). CLBL-1 cells were sensitive to proteasome inhibition by bortezomib and ixazomib. The IC50 of bortezomib was 15.1 nM and of ixazomib was 59.14 nM. Proteasome inhibitors plus doxorubicin had a synergistic effect on CLBL-1 viability; proteosome inhibitors plus vincristine showed different effects depending on the combination ratio, and there was an antagonistic effect with 4-HC. These results may have clinical utility, as proteasome inhibition could potentially be used with a synergizing CHOP compound to improve responsiveness to chemotherapy for canine lymphoma patients.

The pharmacodynamic and mechanistic foundation for the antineoplastic effects of GFH009, a potent and highly selective CDK9 inhibitor for the treatment of hematologic malignancies.

To evade cell cycle controls, malignant cells rely upon rapid expression of select proteins to mitigate proapoptotic signals resulting from damage caused by both cancer treatments and unchecked over-proliferation. Cyclin-dependent kinase 9 (CDK9)-dependent signaling induces transcription of downstream oncogenes promoting tumor growth, especially in hyperproliferative 'oncogene-addicted' cancers, such as human hematological malignancies (HHMs). GFH009, a potent, highly selective CDK9 small molecule inhibitor, demonstrated antiproliferative activity in assorted HHM-derived cell lines, inducing apoptosis at IC50 values below 0.2 μM in 7/10 lines tested. GFH009 inhibited tumor growth at all doses compared to controls and induced apoptosis in a dose-dependent manner. Twice-weekly injections of GFH009 maleate at 10 mg/kg significantly prolonged the survival of MV-4-11 xenograft-bearing rodents, while their body weight remained stable. There was marked reduction of MCL-1 and c-MYC protein expression post-drug exposure both in vitro and in vivo. Through rapid 'on-off' CDK9 inhibition, GFH009 exerts a proapoptotic effect on HHM preclinical models triggered by dynamic deprivation of crucial cell survival signals. Our results mechanistically establish CDK9 as a targetable vulnerability in assorted HHMs and, along with the previously shown superior class kinome selectivity of GFH009 vs other CDK9 inhibitors, strongly support the rationale for currently ongoing clinical studies with this agent in acute myeloid leukemia and other HHMs.

The TiHoCL panel for canine lymphoma: a feasibility study integrating functional genomics and network biology approaches for comparative oncology targeted NGS panel design.

Targeted next-generation sequencing (NGS) enables the identification of genomic variants in cancer patients with high sensitivity at relatively low costs, and has thus opened the era to personalized human oncology. Veterinary medicine tends to adopt new technologies at a slower pace compared to human medicine due to lower funding, nonetheless it embraces technological advancements over time. Hence, it is reasonable to assume that targeted NGS will be incorporated into routine veterinary practice in the foreseeable future. Many animal diseases have well-researched human counterparts and hence, insights gained from the latter might, in principle, be harnessed to elucidate the former. Here, we present the TiHoCL targeted NGS panel as a proof of concept, exemplifying how functional genomics and network approaches can be effectively used to leverage the wealth of information available for human diseases in the development of targeted sequencing panels for veterinary medicine. Specifically, the TiHoCL targeted NGS panel is a molecular tool for characterizing and stratifying canine lymphoma (CL) patients designed based on human non-Hodgkin lymphoma (NHL) research outputs. While various single nucleotide polymorphisms (SNPs) have been associated with high risk of developing NHL, poor prognosis and resistance to treatment in NHL patients, little is known about the genetics of CL. Thus, the ~100 SNPs featured in the TiHoCL targeted NGS panel were selected using functional genomics and network approaches following a literature and database search that shielded ~500 SNPs associated with, in nearly all cases, human hematologic malignancies. The TiHoCL targeted NGS panel underwent technical validation and preliminary functional assessment by sequencing DNA samples isolated from blood of 29 lymphoma dogs using an Ion Torrent™ PGM System achieving good sequencing run metrics. Our design framework holds new possibilities for the design of similar molecular tools applied to other diseases for which limited knowledge is available and will improve drug target discovery and patient care.

Genomic Characterization of Newly Diagnosed Acute Myeloid Leukemia in Patients Age 60 Years and Older; A Report from the Beat AML Master Trial

Background. Acute myeloid leukemia (AML) is a heterogenous disease that occurs primarily in older adults with a median age of 69 years at diagnosis. Most studies that characterize the genomic landscape of AML include younger adults with the majority of patients being younger than 60 years of age. We aimed to study the mutational landscape of newly diagnosed AML patients aged 60 years or older. Methods.Samples were collected from the multi-center Beat AML Master Trial (Burd, Nat Med 2020). Patients were enrolled between 2016 and 2023. Informed consent was obtained according to the Declaration of Helsinki. Patients diagnosed with non-APL AML and who were aged ≥ 60 years old were included. Patients underwent bone marrow aspiration and biopsy, cytogenetic analysis, FLT3-ITD ratio assessment (LeukoStrat CDx FLT3 Mutation Assay, Invivoscribe) and next generation sequencing (NGS) using FoundationOne®Heme(Foundation Medicine) to interrogate the entire coding region of 406 genes and select introns of 31 genes involved in rearrangements, as well as RNA sequencing to interrogate 265 genes known to be somatically altered in human hematological malignancies. The presence of multiple hotspot mutations within one gene in an individual patient was counted as one gene mutation. Patterns of co-occurrence between gene mutations were calculated using the Phi correlation coefficient. Statistical analyses were performed using R statistical software. P<0.01 was considered statistically significant. Results. A total of 1088 newly diagnosed AML patients were identified, of whom 1032 were 60 years or older. Patients had a median age of 72 years at time of diagnosis (range 60-92 years). Forty-two percent of the patients were female, most patients were non-Hispanic and 79% were Caucasian. NGS data were available for 1024 patients and mutations were identified in 461 different genes. Patients had a median number of 11.0 mutations (range 3.0 to 28.0). The most frequently mutated genes were DNMT3A (n=257; 25.1%), TP53(n=255; 24.9%), TET2 (n=243; 23.7%), RUNX1 (n=225; 22.0%), SRSF2 (n=221; 21.6%), ASXL1 (n=216; 21.1%), NPM1 (n=206; 20.1%), FLT3 (n=205; 20.0%), IDH2 (n=190; 18.6%), NRAS (n=208; 16.9%), PTPN11(n=104; 10.2%), STAG2 (n=101, 9.9% and IDH1 (n=99; 9.7%) (Figure 1A). FLT3-ITD was present in 98 (12.1%) of the 810 patients who were tested. Strongest significant co-occurrence was found between FLT3and NPM1 (n=103), STAG2 and ASXL1 (n=63), ASXL1 and RUNX1 (n=92), ASXL1 and SRSF2 (n=88), RUNX1 and SRSF2 (n=94), STAG2 and SRSF2 (n=51) and DNMT3A and NPM1 (n=92) (p<0.01). Pairwise negative correlations were observed between TP53 and NPM1 (n=6), TP53 and SRSF2 (n=17), and between NPM1 and RUNX1 (n=6) (p<0.01) (Figure 1B). The cytogenetic profiles and the full genomic landscape will be presented during the ASH meeting. Discussion. This is the largest study investigating the mutational profile in older adults with AML aged 60 years and older. Due to the 7-day screening period of the Beat AML study, patient selection was biased towards less-rapidly proliferative AML. We showed that the pattern of gene mutations was markedly different from other studies in AML that mainly included younger AML patients (Papaemmanuil, NEJM 2016; TCGA, NEJM 2013 ). The frequency of mutated TP53 was substantially higher in older AML patients, as was the occurrence of myelodysplasia-related gene mutations (e.g., SRSF2, RUNX1, ASXL1, STAG2, U2AF1, and SF3B1) and gene mutations in IDH1, IDH2 and TET2. In contrast, NPM1 mutation was less frequently mutated in older AML patients. Furthermore, we demonstrated that a large proportion (46%) of the AML patients older than 60 years of age do have a mutation that is potentially targetable for treatment ( FLT3 IDH1 IDH2 and NPM1). The high incidence of mutated TP53 and MDS-related gene mutations underscores the increased incidence of high-risk AML and the need for innovative personalized therapies in this older age group. Figure 1. A. Co-occurrence of the 15 most frequently mutated genes, and B. co-correlation between the 15 most frequently mutated genes. Blue indicates positive correlation, red indicated negative correlation. Only significant correlations (p<0.01) are shown.

Effect and Mechanism of NL-101 Combined with Bcl-2 Inhibitor Venetoclax in Acute Myeloid Leukemia

Background Acute myeloid leukemia (AML) is a kind of malignant clonal disease originating from hematopoietic stem cells with strong heterogeneity,with the highly recurrence rate and mortality rate.Although the application of hematopoietic stem cell transplantation and new targeted drugs has improved the prognosis of some patients in recent years, chemotherapy is still the most important treatment for AML. 50-80% of AML patients eventually relapsed after CR, with a low remission rate after relapse.There are reports that BCL-2 overexpression is a clinical feature of human hematologic malignancies that exacerbates the malignant state and drives apoptosis resistance phenotypes.Therefore, how to reduce treatment-related toxic and side effects and overcome tumor cell drug resistance is a hot and difficult topic in current AML treatment. Venetoclax (ABT-199) is an oral, selective Bcl-2 inhibitor. It showed strong anti-leukemia activity in preclinical stage, but its clinical efficacy as a single drug for AML was limited.Nl-101 (also known as EDE-S101)is a new small molecule compound formed by covalently linking the DNA damage core group of Bendamustine hydrochloride with the inhibition core group of histone deacetylase of the histone inhibitor Volinostat (also known as SAHA).Our previous study found that NL-101 single drug can reduce tumor load and prolong survival of AML mice in vivo.In this study, we used Venetoclax in combination with NL-101 to understand its efficacy and safety in vitro and in vivo, and to explore the synergies and mechanisms of action between the two drugs, providing new effective options for the treatment of AML patients. Purpose To explore the efficacy of Bcl-2 inhibitor Venetoclax (ABT-199) and a novel small molecule compound NL-101 in the treatment of acute myeloid leukemia（AML） and the related mechanisms. Experimental Design AML cell lines and primary patient samples were collected and used for cell proliferation assay. MTS colorimetric assay was used to determine the effect of Venetoclax alone or in combination with NL-101 on the growth of AML cells，the combination index (CI) was calculated to evaluate the synergistic effect of the two drugs. Cell death induced by Venetoclax alone or in combination with NL-101 was analyzed by Annexin V-FITC/ Propyl -iodide staining and flow cytometry. Western blot was used to analyze the protein expression levels of MAPK/ERK, PI3K/AK/GSK pathway members and BCL-2 family members.The MV4-11-derived xenograft mouse model was used to assess in vivo efficacy of the combination of venetoclax and NL-101. Results Venetoclax alone and in combination with NL-101 can inhibit the growth of AML cell lines and primary cells to a certain extent, and the combination of drugs has a synergistic effect. Venetoclax combined with NL-101 effectively synergistically induced apoptosis in AML cell lines and primary patient samples. Venetoclax combined with NL-101 can down-regulate the expression of pro-survival proteins in the Bcl-2 family, such as McL-1, Bcl-2, and Bcl-XL, and up-regulate the expression of pro-apoptotic proteins, such as BIM, BID, and BAX/BAK. Phosphorylated ERK, AKT, and JNK expressions were eliminated by venetoclax in combination with NL-101. In vivo results suggest that Venetoclax combined with NL-101 has the potential to treat AML. Conclusions Venetoclax combined with NL-101 can synergistically inhibit the growth and promote apoptosis of AML cell lines and primary AML cells. In vivo experiments demonstrated that Venetoclax combined with NL-101 can synergistically reduce tumor load and prolong survival time in AML mice. These results suggest that Venetoclax in combination with NL-101 is effective in the treatment of acute myeloid leukemia.

Mesenchymal Stem Cells‐Derived Extracellular Vesicles in Orthopedic Diseases: Recent Advances and Therapeutic Potential

AbstractEver since the first application of mesenchymal stem cell (MSC) transplantation treating human hematologic malignancies in 1995, MSC‐based treatments have demonstrated great therapeutic potential in clinical settings. However, only a few MSC‐based cell therapy products have been clinically approved. Accumulating evidence suggests that the beneficial effects of MSCs are mainly attributed to the release of paracrine factors or extracellular vesicles (EVs) rather than their mesodermal differentiation potential. Therefore, MSC‐derived EVs (MSC‐EVs), such as exosomes and microvesicles, have merged as promising alternatives to traditional cell‐based therapeutics in clinical practice. They offer several advantages such as better safety, lower immunogenicity, protection of cargoes from degradation, and the ability to overcome biological barriers. Moreover, there have been multiple clinical studies exploring the potential of MSC‐EVs for treating various diseases, including orthopedic disorders. However, there is no definitive “cure” for conditions such as osteoporosis and other bone disorders, but MSC‐EVs have displayed significant therapeutic potential for these orthopedic ailments. Therefore, the objective of this study is to conduct a systematic review of current knowledge related to MSC‐EVs and emphasize their potential application in treating orthopedic diseases, such as bone defects, osteoarthritis, osteoporosis, intervertebral disc degeneration, osteosarcoma, and osteoradionecrosis.

Progress in construction of mouse models to investigate the pathogenesis and immune therapy of human hematological malignancy.

Hematological malignancy is a disease arisen by complicate reasons that seriously endangers human health. The research on its pathogenesis and therapies depends on the usage of animal models. Conventional animal model cannot faithfully mirror some characteristics of human features due to the evolutionary divergence, whereas the mouse models hosting human hematological malignancy are more and more applied in basic as well as translational investigations in recent years. According to the construction methods, they can be divided into different types (e.g. cell-derived xenograft (CDX) and patient-derived xenograft model (PDX) model) that have diverse characteristics and application values. In addition, a variety of strategies have been developed to improve human hematological malignant cell engraftment and differentiation in vivo. Moreover, the humanized mouse model with both functional human immune system and autologous human hematological malignancy provides a unique tool for the evaluation of the efficacy of novel immunotherapeutic drugs/approaches. Herein, we first review the evolution of the mouse model of human hematological malignancy; Then, we analyze the characteristics of different types of models and summarize the ways to improve the models; Finally, the way and value of humanized mouse model of human immune system in the immunotherapy of human hematological malignancy are discussed.

TOX2 coordinates with TET2 to positively regulate central memory differentiation in human CAR T cells.

Chimeric antigen receptor (CAR) T cell therapy is used in treating human hematological malignancies, but its efficacy is limited by T cell exhaustion (TEX). TEX arises at the expense of central memory T cells (TCM), which exhibit robust antitumor efficacy. Reduction of the TET2 gene led to increased TCM differentiation in a patient with leukemia who experienced a complete remission. We show that loss of TET2 led to increased chromatin accessibility at exhaustion regulators TOX and TOX2, plus increased expression of TOX2. Knockdown of TOX increased the percentage of TCM. However, unexpectedly, knockdown of TOX2 decreased TCM percentage and reduced proliferation. Consistently, a TCM gene signature was reduced in the TOX2 knockdown, and TOX2 bound to promoters of numerous TCM genes. Our results thus suggest a role for human TOX2, in contrast to exhaustion regulator TOX, as a potentiator of central memory differentiation of CAR T cells, with plausible utility in CAR T cell cancer therapy via modulated TOX2 expression.

Discovery of N-(2-chloro-5-(3-(pyridin-4-yl)-1H-pyrazolo[3,4-b]pyridin-5-yl)pyridin-3-yl)-4-fluorobenzenesulfonamide (FD274) as a highly potent PI3K/mTOR dual inhibitor for the treatment of acute myeloid leukemia

PI3K-Akt-mTOR pathway is a highly activated signal transduction pathway in human hematological malignancies and has been validated as a promising target for acute myeloid leukemia (AML) therapy. Herein, we designed and synthesized a series of 7-azaindazole derivatives as potent PI3K/mTOR dual inhibitors based on our previously reported FD223. Among them, compound FD274 showed excellent dual PI3K/mTOR inhibitory activity, with IC50 values against PI3Kα/β/γ/δ and mTOR of 0.65 nM, 1.57 nM, 0.65 nM, 0.42 nM, and 2.03 nM, respectively, superior to compound FD223. Compared to the positive drug Dactolisib, FD274 exhibited significant anti-proliferation of AML cell lines (HL-60 and MOLM-16 with IC50 values of 0.092 μM and 0.084 μM, respectively) in vitro. Furthermore, FD274 demonstrated dose-dependent inhibition of tumor growth in the HL-60 xenograft model in vivo, with 91% inhibition of tumor growth at an intraperitoneal injection dose of 10 mg/kg and no observable toxicity. All of these results suggest that FD274 has potential for further development as a promising PI3K/mTOR targeted anti-AML drug candidate.

Abstract 5654: A therapeutic humanized anti-carcinoma monoclonal antibody (mAb) NEO-201 can also target human granulocytic myeloid-derived suppressor cells (gMDSCs) and regulatory T (Tregs) cells

Abstract Background: NEO-201 is a humanized IgG1 mAb reactive against multiple human cancers but not against most normal epithelial tissues. NEO-201 binds to core 1 or extended core 1 O-glycans expressed by its target cells, including neutrophils, various carcinomas, and some human hematological malignancies. NEO-201 can mediate antitumor activity through antibody-dependent cellular cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), and blockade of the CEACAM5/CEACAM1 ICI pathway. A previous study using flow cytometry demonstrated that NEO-201+/CD4+ T cells were also CD25+/CD127−/Foxp3+/CD15s+ using PBMCs from healthy donors (HD). NEO-201 can kill these Treg cells through CDC in vitro. NEO-201 does not bind to the majority of CD4+ T cells and to other immune subsets. Human gMDSCs are increased in cancer patients and are a population of immature MDSCs deriving from immature neutrophils and alternative activation of mature neutrophils. gMDSC are characterized by HLA-DR−, CD11b+, CD33+, CD15+phenotype.We have shown that NEO-201 recognizes and kill human neutrophils through ADCC. This current investigation was designed to evaluate whether NEO-201 can target and mediate ADCC activity against human gMDSCs. Methods: gMDSCs were generated from human neutrophils from 5 HD isolated using EasySepTM direct human neutrophil isolation kit. Isolated neutrophils were cultured in complete RPMI1640 medium supplemented with human GM-CSF and human IL-6 for 7 days. Phenotypic analysis by flow cytometry was performed on the generated gMDSCs using NEO-201 and mAbs against human CD33, HLA-DR, CD15, CD14, CD66b. Flow cytometry based ADCC assay was performed using gMDSCs stained with both CD33 and HLA-DR as target. PBMCs from a separate HD were used as effectors at different E:T ratios. The ADCC activity of NEO-201 was evaluated comparing the percentage of CD33+/HLA-DR− viable cells in gMDSCs incubated with medium alone to the percentage of CD33+/HLA-DR− viable cells incubated with PBMCs alone and with PBMCs plus NEO-201. Results: Flow cytometry analysis revealed that gMDSCs can be generated from human neutrophils after 7 days of culture with GM-CSF and IL-6 and that they express the following phenotype: HLA-DR−/CD33+/CD15+/CD14−/CD66b+. NEO-201 bound to the majority of these gMDSCs. NEO-201 was functional in mediating ADCC to kill these gMDSCs. Conclusion: This study demonstrated that NEO-201 can be used to identify and kill suppressive gMDSCs in addition to Treg cells. Depletion of suppressive Tregs and gMDSCs in the TME could be an effective strategy to prevent hyperprogressive disease when anti-PD-1 is used in cancer immunotherapy. These data support the rationale for the ongoing phase II clinical trial using NEO-201 in combination with pembrolizumab in checkpoint refractory patients with metastatic solid tumors. Citation Format: Massimo Fantini, Christina M. Annunziata, Philip M. Arlen, Kwong Y. Tsang. A therapeutic humanized anti-carcinoma monoclonal antibody (mAb) NEO-201 can also target human granulocytic myeloid-derived suppressor cells (gMDSCs) and regulatory T (Tregs) cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5654.

Coronavirus Disease 2019-Associated Pulmonary Aspergillosis: A Noninvasive Screening Model for Additional Diagnostics.

Coronavirus disease 2019 (COVID-19)-associated pulmonary aspergillosis (CAPA) is likely underdiagnosed, and current diagnostic tools are either invasive or insensitive. A retrospective study of mechanically ventilated patients with COVID-19 admitted to 5 Johns Hopkins hospitals between March 2020 and June 2021 was performed. Multivariable logistic regression was used for the CAPA prediction model building. Performance of the model was assessed using the area under the receiver operating characteristic curve (AUC). In the cohort of 832 patients, 98 (11.8%) met criteria for CAPA. Age, time since intubation, dexamethasone for COVID-19 treatment, underlying pulmonary circulatory diseases, human immunodeficiency virus, multiple myeloma, cancer, or hematologic malignancies were statistically significantly associated with CAPA and were included in the CAPA prediction model, which showed an AUC of 0.75 (95% confidence interval, .70-.80). At a screening cutoff of ≥0.085, it had a sensitivity of 82%, a specificity of 51%, a positive predictive value of 18.6%, and a negative predictive value of 95.3%. (The CAPA screening score calculator is available at www.transplantmodels.com). We developed a CAPA risk score as a noninvasive tool to aid in CAPA screening for patients with severe COVID-19. Our score will also identify a group of patients who are unlikely to have CAPA and who therefore need not undergo additional diagnostics and/or empiric antifungal therapy.

Pediatric Preclinical Testing Consortium Evaluation of the MCL1 Inhibitor, AMG 176, Against Xenograft Models of Acute Lymphoblastic Leukemia

Introduction: Children diagnosed with acute lymphoblastic leukemia (ALL) experience close to a 90% likelihood of cure, however the outcome for high-risk subtypes remains poor. Evasion of apoptosis is a hallmark of cancer, with dysregulation of the BCL-2 family of proteins often reported (Delbridge et al, Nat Rev Cancer 16:99-109, 2016). The BCL-2 family consists of the anti-apoptotic MCL1, BCL-2 and BCLXL as well as pro-apoptotic proteins. AMG 176, a first-in-class MCL1 inhibitor, previously demonstrated potent apoptosis-inducing activity in the nanomolar range against cell lines and patient explants of human hematological malignancies (Caenepeel et al, Cancer Discov 8:1582-97, 2018). Furthermore, discontinuous oral administration of AMG 176 inhibited the growth of human acute myeloid leukemia and multiple myeloma tumor xenografts in vivo at well-tolerated doses. AMG 176 has entered clinical evaluation for patients with hematological malignancies. Therefore, it was of interest for the Pediatric Preclinical Testing Consortium (PPTC) to evaluate the in vivo efficacy of AMG 176 against its patient-derived xenograft (PDX) models of pediatric ALL. Methods:MCL1, BCL-2 and BCLXL mRNA expression was quantified by RNA-seq (http://pedcbioportal.kidsfirstdrc.org). For in vitro cytotoxicity assays, cells were treated with AMG 176 (10 μM - 1 pM) or DMSO control for 24 h and cell viability was determined by AlamarBlue assay and expressed as IC50values. AMG 176 was evaluated in vivo against a panel of 38 ALL PDXs in a single mouse trial format. Each PDX was inoculated via tail vein injection into one NSG mouse (1-5 x 106 cells per mouse). Engraftment and drug responses were determined by enumerating the proportion of human versus mouse CD45+ (%huCD45+) cells in the peripheral blood (PB) at weekly intervals. Treatment with AMG 176 (provided by Amgen, Inc.) began once the %huCD45+ was ≥1% in each mouse and consisted of 60 mg/kg via oral gavage 2 days on, 5 days off for 2 weeks. The experimental endpoint was pre-defined as the %huCD45+ reaching 25% in the PB. Drug efficacy was assessed by event-free survival (EFS) and stringent objective response measures (Houghton et al, Pediatr Blood Cancer 49:928-40, 2007). For the pharmacokinetic (PK) study naïve NSG mice were treated with either vehicle or AMG 176 (60 mg/kg) and groups of 3 mice harvested up to 24 h post-treatment. Plasma samples were collected for analysis of AMG 176 using acetonitrile protein precipitation and LC-MS/MS with gradient elution on a Phenomenex Kinetex XB-C18 (3.0 x 50 mm, 2.6 µm) analytical column. Results:MCL1 mRNA expression ranged from 21-282 FPKM across a panel of 90 ALL PDXs representing 6 broad ALL subtypes (B-cell precursor, BCP-ALL; Ph+-ALL; Ph-like ALL; mixed-lineage leukemia-rearranged, MLLr-ALL; T-cell ALL, T-ALL; and early T-cell precursor ALL, ETP-ALL). Thirty eight of the 90 ALL PDXs were selected for in vivo evaluation of AMG 176 activity based on their profiles of MCL1, BCL-2 and BCLXL mRNA expression. AMG 176 was well tolerated in the efficacy study, with only 3 mice experiencing >10% weight loss. Mouse EFS values ranged from 3.5 to 47.8 days from treatment initiation, however no PDX achieved an objective response (remission) with all but one PDX exhibiting progressive disease. The Ph-like ALL PDX, ALL-102 (MCL1 expression 71.8 FPKM) scored a stable disease and had the highest EFS value (47.8 days). In an effort to explain the poor in vivo activity of AMG 176 several follow-up studies were performed. First, the in vitro IC50 values elicited by the AMG-176 stock against 5 leukemia cell lines (MOLM-13, 1.6 µM; NB-4, 2.0 µM; Kasumi-1, 1.6 µM; THP-1, 1.2 µM; and U-937,1.3 µM) were comparable to published data. Second, C18 LC-MS analysis revealed major and minor peaks consistent with theoretical peaks with negligible degradation. Third, PK study results demonstrated that the AMG 176 plasma concentration peaked at 8.5 µM, one hour after administration, with an AUC of 87.7 µM*hr and t1/2 of 4.1 h. This AUC was markedly lower than that in athymic nude mice treated at the same dose (1,120 µM*hr). Conclusions: AMG 176 exhibited modest in vivo activity against a large range of pediatric ALL PDXs, with the best response defined as stable disease in a single PDX. PK data suggest that AMG 176 systemic exposures in NSG mice may not be sufficient to elicit meaningful and prolonged regressions in pediatric ALL PDXs. (Supported by NCI Grants CA199000 and CA199222)

37-Year-Old Man With Headache and Nasal Congestion

37-Year-Old Man With Headache and Nasal Congestion

PB2012: A PHASE 2 MULTIARM STUDY OF MAGROLIMAB COMBINATIONS IN PATIENTS WITH RELAPSED/REFRACTORY MULTIPLE MYELOMA

Background: Magrolimab is a first-in-class humanized monoclonal antibody that blocks the immune checkpoint CD47, a “do not eat me” signal, overexpressed on tumor cells. Binding of magrolimab to CD47 leads to phagocytosis of cancer cells in human solid tumors and hematologic malignancies. CD47 is highly expressed in multiple myeloma (MM), and overexpression contributes to MM pathogenesis. Blocking of CD47 induces immediate activation of macrophages and elimination of myeloma cells. Hence, a blockade of CD47 with magrolimab may have therapeutic benefit in patients with MM. Additionally, in vitro studies have shown synergy between magrolimab and daratumumab in MM cell line phagocytosis, and complimentary mechanisms suggest potential enhanced efficacy in other combinations. Aims: To evaluate the safety, tolerability, and efficacy of magrolimab in combination with commonly used myeloma therapies in patients with relapsed/refractory MM (RRMM). Methods: This phase 2 open-label multiarm study includes patients aged ≥18 years currently requiring treatment for RRMM; all participants must have received ≥3 previous lines of therapy for MM, including an immunomodulatory drug and a proteasome inhibitor. This study includes safety run-in and corresponding dose-expansion phases with the following combinations: magrolimab + daratumumab, magrolimab + pomalidomide + dexamethasone, magrolimab + carfilzomib + dexamethasone, and magrolimab + bortezomib + dexamethasone (Figure). Magrolimab + bortezomib + dexamethasone may be initiated based on the preliminary safety and efficacy of magrolimab + carfilzomib + dexamethasone, and, if initiated, only requires 1 prior line of therapy. The primary objectives of the safety run-in phase are to evaluate the safety and tolerability of magrolimab and determine the recommended phase 2 dose (RP2D), with incidence of dose-limiting toxicities (DLTs), adverse events, and laboratory abnormalities as the primary endpoint. The dose-expansion phase will evaluate efficacy, with objective response rate as the primary endpoint. Secondary endpoints include duration of response, progression-free survival, and overall survival. Minimal residual disease negativity, biomarker changes from baseline, immune cell signaling alterations, and the mutational profile of myeloma cells will also be explored. Magrolimab will be administered intravenously as an initial priming dose to mitigate on-target anemia, followed by a maintenance dose on days 8, 15, 22, and 29 during cycle 1 (35 days); days 1, 8, 15, and 22 during cycle 2 (28 days); and days 1 and 15 from cycle 3 (28 days) onward (maximum of 8 cycles in patients who previously received bortezomib). Daratumumab, pomalidomide, dexamethasone, carfilzomib, and bortezomib will be administered according to manufacturer’s prescribing information. Dose de-escalation may occur based on DLTs per protocol in the safety run-in cohorts; magrolimab doses will not be reduced in the dose-expansion cohorts. Patients will continue treatment until unacceptable toxicity, progressive disease, or patient/investigator choice to discontinue. Planned enrollment is approximately 153 patients, with up to 27 in each safety run-in cohort and 72 in the dose-expansion cohorts following completion of the safety run-ins. Results: Trial in progress. Image:Summary/Conclusion: Patient recruitment is ongoing. Clinical trial information: NCT04892446.

Optimized flow cytometric detection of transient receptor potential vanilloid-1 (TRPV1) in human hematological malignancies

The ectopic overexpression of transient receptor potential vanilloid-1 (TRPV1) has been detected in numerous solid cancers, including breast, prostate, pancreatic, and tongue epithelium cancer. However, the expression of TRPV1 in hematological malignancies remains unknown. Here we show through in silico analysis that elevated TRPV1 mRNA expression occurs in a range of hematological malignancies and presents an optimized flow cytometry method to rapidly assess TRPV1 protein expression for both cell lines and primary patient samples. Three anti-TRPV1 antibodies were evaluated for intracellular TRPV1 detection using flow cytometry resulting in an optimized protocol for the evaluation of TRPV1 in hematological malignant cell lines and patients’ peripheral blood mononuclear cells (PBMC). Overexpression of TRPV1 was observed in THP-1 (acute monocytic leukemia) and U266B1 (multiple myeloma, MM), but not U937 (histiocytic lymphoma) compared to healthy PBMC. TRPV1 was also detected in all 49 patients including B-cell non-Hodgkin’s lymphoma (B-NHL), MM, and others and 20 healthy controls. TRPV1 expression was increased in 8% of patients (MM = 2, B-NHL = 2). In conclusion, we provide an optimized flow cytometry method for routine expression analysis of clinical samples and show that TRPV1 is increased in a subset of patients with hematological malignancies.

Facial demodicosis in the immunosuppressed state: a retrospective case series from a tertiary referral center.

Data on Demodex in the immunosuppressed state is limited, focusing mainly on patients with human immunodeficiency virus and hematological malignancies. The aim of this study was to describe the manifestations of facial demodicosis in diverse immunosuppressive states. The medical records of all patients followed at a Demodex outpatient clinic of a tertiary medical center from January 2008 to November 2020 were retrospectively reviewed. Data on patients who were immunosuppressed while with demodicosis were retrieved. The cohort included 28 patients (17 women and 11 men; median age, 58 years). Types of immunosuppression included treatments with hydroxyurea for polycythemia vera/essential thrombocytosis, mycophenolic acid, tacrolimus, and prednisone for liver and/or kidney transplantation, prednisone with cyclosporine/methotrexate/azathioprine/rituximab mainly for autoimmune diseases, mercaptopurine with/without anti-tumor necrosis factor alpha (TNF-α) for Crohn's disease, chemotherapy for neoplasms, anti-TNF-α for psoriasis, and Cushing's syndrome. The clinical types of demodicosis included: papulopustular, erythematotelangiectatic and fulminant rosacea, hyperpigmented, pityriasis folliculorum, pustular folliculitis, and dermatitis. The diverse clinical presentations led to various differential diagnoses. Topical treatment with ivermectin (monotherapy/combination with other treatments) was effective. Clinicians treating immunosuppressed patients should be familiar with the different forms of demodicosis and include them in the differential diagnosis of facial eruptions.