CAR T cells targeting options in the fight against multiple myeloma.

INTRODUCTION: Multiple myeloma (MM) is a hematological malignancy in which patients present with bone marrow infiltration of clonal terminally-differentiated plasma cells. Monoclonal protein in the serum and/or urine is frequently detected. Over the past decade, important progress has been made in the comprehension of disease biology and treatment personalization. Much work has been put into the development of chimeric antigen receptor (CAR) gene-modified T-cell therapy thought to be a promising therapeutic option for pluritreated patients with refractory MM. EVIDENCE ACQUISITION: We performed an analysis of clinical trials registered at the international repository clinicaltrials.gov using "CAR" OR "CAR T" AND "multiple myeloma" as search terms to understand what were the antigens targeted by CAR T strategies and what was the trade-off of their exploitation. The search retrieved a list of 103 trials that was manually filtered to eliminate follow-up and observational or not-pertinent trials. EVIDENCE SYNTHESIS: Most studies employed anti-BCMA targeting either alone (62/94; 66%), or in combination with a second target (12/94; 13%). The second target most studied was SLAMF7 (CD319) explored by 4/94 (4%) clinical trials. Other antigens investigated and described here include: CD44v6, CD38, CD138, MUC1, CD56, CD19, Igk light chain, Lewis Y, CD229 and GPRC5D. CONCLUSIONS: Targeting an appropriate antigen(s) is the key to both safety and efficacy of CAR T approaches in MM as there is dearth of tumor-specific antigens. Most antigens tested are merely enriched on MM cells. Working with tumor-enriched antigens requires careful assessment of the balance between harm (toxicity) and benefit (disease eradication) to the patient. This review provides an up-to-date overview of the avenues that are being explored.

A Novel Platform to Test In Vivo Single Gene Dependencies in t(8,21) and t(15,17) AML Confirms Zeb2 as Leukemia Target.

The increased usage of high-throughput technologies in cancer research, including genetic and drug screens, generates large sets of candidate targets that need to be functionally validated for their roles in tumor development. Thus, reliable and robust in vivo model systems are needed to perform reverse genetic experiments. Ideally, these models should allow for a conditional silencing of the target and an unambiguous identification of engineered cancer cells. Here, we present a platform consisting of: (i) t(8;21) and t(15;17) driven acute myeloid leukemia (AML) transgenic mice with constitutive expression of green fluorescent protein (GFP) and inducible expression of Cre recombinase, and (ii) REX, a modified pSico lentiviral vector for inducible shRNA expression and red fluorescent protein (RFP) as a selection marker. In this system, leukemic cells from transgenic mice are transduced with REX, flow sorted, and transplanted into syngeneic hosts. Gene interference is induced in established tumors by tamoxifen treatment. Dual-color cell fluorescence guides the in vivo identification of shRNA interfered AML cells, monitoring engraftment and disease progression. We tested the platform by inducing knockdown of Zeb2, a gene upregulated by AML1-ETO and PML-RARα oncogenes in pre-leukemic hematopoietic stem cell compartment, and observed a significant delay in leukemia onset. This proves the power and utility of the platform and confirms Zeb2 contribution to the pathogenesis of AML.

HDAC8: A Promising Therapeutic Target for Acute Myeloid Leukemia.

Histone deacetylase 8 (HDAC8), a class I HDAC that modifies non-histone proteins such as p53, is highly expressed in different hematological neoplasms including a subtype of acute myeloid leukemia (AML) bearing inversion of chromosome 16 [inv(16)]. To investigate HDAC8 contribution to hematopoietic stem cell maintenance and myeloid leukemic transformation, we generated a zebrafish model with Hdac8 overexpression and observed an increase in hematopoietic stem/progenitor cells, a phenotype that could be reverted using a specific HDAC8 inhibitor, PCI-34051 (PCI). In addition, we demonstrated that AML cell lines respond differently to PCI treatment: HDAC8 inhibition elicits cytotoxic effect with cell cycle arrest followed by apoptosis in THP-1 cells, and cytostatic effect in HL60 cells that lack p53. A combination of cytarabine, a standard anti-AML chemotherapeutic, with PCI resulted in a synergistic effect in all the cell lines tested. We, then, searched for a mechanism behind cell cycle arrest caused by HDAC8 inhibition in the absence of functional p53 and demonstrated an involvement of the canonical WNT signaling in zebrafish and in cell lines. Together, we provide the evidence for the role of HDAC8 in hematopoietic stem cell differentiation in zebrafish and AML cell lines, suggesting HDAC8 inhibition as a therapeutic target in hematological malignancies. Accordingly, we demonstrated the utility of a highly specific HDAC8 inhibition as a therapeutic strategy in combination with standard chemotherapy.

Has Drug Repurposing Fulfilled its Promise in Acute Myeloid Leukaemia?

Drug repurposing is a method of drug discovery that consists of finding a new therapeutic context for an old drug. Compound identification arises from screening of large libraries of active compounds, through interrogating databases of cell line gene expression response upon treatment or by merging several types of information concerning disease-drug relationships. Although, there is a general consensus on the potential and advantages of this drug discovery modality, at the practical level to-date no non-anti-cancer repurposed compounds have been introduced into standard acute myeloid leukaemia (AML) management, albeit that preclinical validation yielded several candidates. The review presents the state-of-the-art drug repurposing approach in AML and poses the question of what has to be done in order to take a full advantage of it, both at the stage of screening design and later when progressing from the preclinical to the clinical phases of drug development. We argue that improvements are needed to model and read-out systems as well as to screening technologies, but also to more funding and trust in drug repurposing strategies.

Wnt Signalling in Acute Myeloid Leukaemia.

Acute myeloid leukaemia (AML) is a group of malignant diseases of the haematopoietic system. AML occurs as the result of mutations in haematopoietic stem/progenitor cells, which upregulate Wnt signalling through a variety of mechanisms. Other mechanisms of Wnt activation in AML have been described such as Wnt antagonist inactivation through promoter methylation. Wnt signalling is necessary for the maintenance of leukaemic stem cells. Several molecules involved in or modulating Wnt signalling have a prognostic value in AML. These include: ß-catenin, LEF-1, phosphorylated-GSK3ß, PSMD2, PPARD, XPNPEP, sFRP2, RUNX1, AXIN2, PCDH17, CXXC5, LLGL1 and PTK7. Targeting Wnt signalling for tumour eradication is an approach that is being explored in haematological and solid tumours. A number of preclinical studies confirms its feasibility, albeit, so far no reliable clinical trial data are available to prove its utility and efficacy.

Consolidation with 90Y ibritumomab tiuxetan radioimmunotherapy in mantle cell lymphoma patients ineligible for high dose therapy: results of the phase II multicentre Polish Lymphoma Research Group trial, after 8-year long follow-up.

Polish Lymphoma Research Group performed a phase-II trial to test whether 90Y ibritumomab tiuxetan radioimmunotherapy (Y90) may constitute an alternative consolidation for mantle cell lymphoma patients unfit for high-dose therapy. Forty-six patients were consolidated with Y90 following response to the 1st (n = 34) or 2nd line (n = 12) (immuno)chemotherapy. Majority of the patients had advanced disease (stage IV and presence of B-symptoms in 85% and 70%, respectively) and high MIPI (5.8, range 4-7). Consolidation with Y90 increased the complete remission (CR) rate obtained by the 1st line therapy from 41% to 91% and allowed for median PFS of 3.3 and OS of 6.5 years. In the first relapse, CR rate increased from 16% to 75%, while median PFS and OS totaled 2.2 and 6.5 years, respectively. At 8 years, 30% of patients, consolidated in the 1st line CR were alive, without relapse. Toxicity associated with Y90 is manageable, more severe after fludarabine-based regimens.

Adhesion Deregulation in Acute Myeloid Leukaemia.

Cell adhesion is a process through which cells interact with and attach to neighboring cells or matrix using specialized surface cell adhesion molecules (AMs). Adhesion plays an important role in normal haematopoiesis and in acute myeloid leukaemia (AML). AML blasts express many of the AMs identified on normal haematopoietic precursors. Differential expression of AMs between normal haematopoietic cells and leukaemic blasts has been documented to a variable extent, likely reflecting the heterogeneity of the disease. AMs govern a variety of processes within the bone marrow (BM), such as migration, homing, and quiescence. AML blasts home to the BM, as the AM-mediated interaction with the niche protects them from chemotherapeutic agents. On the contrary, they detach from the niches and move from the BM into the peripheral blood to colonize other sites, i.e., the spleen and liver, possibly in a process that is reminiscent of epithelial-to-mesenchymal-transition in metastatic solid cancers. The expression of AMs has a prognostic impact and there are ongoing efforts to therapeutically target adhesion in the fight against leukaemia.

Primary Cardioprotection Reduces Mortality in Lymphoma Patients with Increased Risk of Anthracycline Cardiotoxicity, Treated by R-CHOP Regimen.

BACKGROUND: Advances in anti-lymphoma therapy prolong overall survival, making late adverse effects, like doxorubicin-related cardiotoxicity, an even more important clinical issue. The effectiveness of cardioprotective strategies with close monitoring, angiotensin-converting enzyme inhibitors and/or ß-blockers as well as liposomal doxorubicin are still unconfirmed in clinical practice. METHODS: This study evaluated the role of a primary cardioprotection strategy in preventing cardiovascular mortality and heart failure occurrence in non-Hodgkin lymphoma (NHL) patients with a high risk of anthracycline cardiotoxicity. Thirty-five NHL patients were subjected prospectively to ramipril and/or bisoprolol at NHL diagnosis, before implementing doxorubicin-containing regimens. Additionally, patients with a diagnosis of asymptomatic/mild heart failure received the liposomal form of doxorubicin. The clinical outcome and frequency of all serious cardiac events were compared with the results in a historical cohort of 62 high-risk cases treated without primary cardioprotection. RESULTS: NHL patients with a primary cardioprotection strategy did not experience cardiovascular deaths in contrast to the retrospective control group where cardiovascular mortality was 14.5% at 3 years (p < 0.05). Primary cardioprotection also decreased the frequency of new cardiotoxicity-related clinical symptoms (2.8 vs. 24.1%; p < 0.05) and prevented the occurrence of cardiac systolic dysfunction (0 vs. 8.5%, respectively; p < 0.05). Although the study was not planned to detect any survival benefit, it demonstrated a trend towards increased response rates (complete response 82 vs. 67%; p not significant) and prolonged survival (projected 5-year overall survival 74 vs. 60%; p < 0.05) for patients treated with primary cardioprotection. CONCLUSIONS: A primary personalized cardioprotection strategy decreases the number of cardiac deaths and may potentially prolong overall survival in NHL patients with increased risk of anthracycline cardiotoxicity.

Understanding the molecular basis of acute myeloid leukemias: where are we now?

Although the treatment modalities for acute myeloid leukemia (AML) have not changed much over the past 40 years, distinct progress has been made in deciphering the basic biology underlying the pathogenesis of this group of hematological disorders. Studies show that AML development is a multicause, multistep and multipathway process. Accordingly, AMLs constitute a heterogeneous group of diseases. The thorough understanding of the molecular basis of AML is paving the way for better therapeutic approaches. Multiple novel drugs are being introduced and new, more efficient and less toxic formulations of conventional therapeutics are becoming available. Here, we review the recent advances in the comprehension of the molecular processes that lead to the onset of AML and its translation into clinical practice.

AML1/ETO accelerates cell migration and impairs cell-to-cell adhesion and homing of hematopoietic stem/progenitor cells.

The AML1/ETO fusion protein found in acute myeloid leukemias functions as a transcriptional regulator by recruiting co-repressor complexes to its DNA binding site. In order to extend the understanding of its role in preleukemia, we expressed AML1/ETO in a murine immortalized pluripotent hematopoietic stem/progenitor cell line, EML C1, and found that genes involved in functions such as cell-to-cell adhesion and cell motility were among the most significantly regulated as determined by RNA sequencing. In functional assays, AML1/ETO-expressing cells showed a decrease in adhesion to stromal cells, an increase of cell migration rate in vitro, and displayed an impairment in homing and engraftment in vivo upon transplantation into recipient mice. Our results suggest that AML1/ETO expression determines a more mobile and less adherent phenotype in preleukemic cells, therefore altering the interaction with the hematopoietic niche, potentially leading to the migration across the bone marrow barrier and to disease progression.

Nucleophosmin leukemogenic mutant activates Wnt signaling during zebrafish development.

Nucleophosmin (NPM1) is a ubiquitous multifunctional phosphoprotein with both oncogenic and tumor suppressor functions. Mutations of the NPM1 gene are the most frequent genetic alterations in acute myeloid leukemia (AML) and result in the expression of a mutant protein with aberrant cytoplasmic localization, NPMc+. Although NPMc+ causes myeloproliferation and AML in animal models, its mechanism of action remains largely unknown. Here we report that NPMc+ activates canonical Wnt signaling during the early phases of zebrafish development and determines a Wnt-dependent increase in the number of progenitor cells during primitive hematopoiesis. Coherently, the canonical Wnt pathway is active in AML blasts bearing NPMc+ and depletion of the mutant protein in the patient derived OCI-AML3 cell line leads to a decrease in the levels of active ß-catenin and of Wnt target genes. Our results reveal a novel function of NPMc+ and provide insight into the molecular pathogenesis of AML bearing NPM1 mutations.

Molecular investigation of coexistent chronic myeloid leukaemia and peripheral T-cell lymphoma - a case report.

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm underlain by the formation of BCR-ABL1 - an aberrant tyrosine kinase - in the leukaemic blasts. Long-term survival rates in CML prior to the advent of tyrosine kinase inhibitors (TKIs) were dismal, albeit the incidence of secondary malignancies was higher than that of age-matched population. Current figures confirm the safety of TKIs with conflicting data concerning the increased risk of secondary tumours. We postulate that care has to be taken when distinguishing between coexisting, secondary-to-treatment and second in sequence, but independent tumourigenic events, in order to achieve an unbiased picture of the adverse effects of novel treatments. To illustrate this point, we present a case of a patient in which CML and peripheral T-cell lymphoma (PTCL) coexisted, although the clinical presentation of the latter followed the achievement of major molecular response of CML to TKIs.

A monoclonal antibody against mutated nucleophosmin 1 for the molecular diagnosis of acute myeloid leukemias.

Mutations in the nucleophosmin 1 (NPM1) gene are the most frequent genetic aberrations of acute myeloid leukemia (AML) and define a clinically distinct subset of AML. A monoclonal antibody (T26) was raised against a 19-amino acid polypeptide containing the unique C-terminus of the type A NPM1 mutant protein. T26 recognized 10 of the 21 known NPM1 mutants, including the A, B, and D types, which cover approximately 95% of all cases, and did not cross-react with wild-type NPM1 or unrelated cellular proteins. It performed efficiently with different detection technologies, including immunofluorescence, immunohistochemistry, and flow cytometry. Within a series of consecutive de novo AML patients, 44 of 110 (40%) and 15 of 39 (38%) cases scored positive using the T26 antibody in immunofluorescence and flow cytometry assays, respectively. T26-positive cases were found to be all carrying mutations of NPM1 exclusively, as determined by molecular analysis. T26 is the first antibody that specifically recognizes a leukemia-associated mutant protein. Immunofluorescence or flow cytometry using T26 may thus become a new tool for a rapid, simple, and cost-effective molecular diagnosis of AMLs.