Tomás-Daza L, Rovirosa L, López-Martí P, Nieto-Aliseda A, Serra F, Planas-Riverola A, Molina O, McDonald R, Ghevaert C, Cuatrecasas E, Costa D, Camós M, Bueno C, Menéndez P, Valencia A, Javierre BM
Low input capture Hi-C (liCHi-C) identifies promoter-enhancer interactions at high-resolution.
Nature Commununications17 Jan 2023, 14(1)268. Epub 17 Jan 2023
Long-range interactions between regulatory elements and promoters are key in gene transcriptional control; however, their study requires large amounts of starting material, which is not compatible with clinical scenarios nor the study of rare cell populations. Here we introduce low input capture Hi-C (liCHi-C) as a cost-effective, flexible method to map and robustly compare promoter interactomes at high resolution. As proof of its broad applicability, we implement liCHi-C to study normal and malignant human hematopoietic hierarchy in clinical samples. We demonstrate that the dynamic promoter architecture identifies developmental trajectories and orchestrates transcriptional transitions during cell-state commitment. Moreover, liCHi-C enables the identification of disease-relevant cell types, genes and pathways potentially deregulated by non-coding alterations at distal regulatory elements. Finally, we show that liCHi-C can be harnessed to uncover genome-wide structural variants, resolve their breakpoints and infer their pathogenic effects. Collectively, our optimized liCHi-C method expands the study of 3D chromatin organization to unique, low-abundance cell populations, and offers an opportunity to uncover factors and regulatory networks involved in disease pathogenesis.
Jiménez-Reinoso A, Tirado N, Martinez-Moreno A, Díaz VM, García-Peydró M, Hangiu O, Díez-Alonso L, Albitre Á, Penela P, Toribio ML, Menéndez P, Álvarez-Vallina L, Sánchez Martínez D
Efficient preclinical treatment of cortical T cell acute lymphoblastic leukemia with T lymphocytes secreting anti-CD1a T cell engagers.
J Immunother CancerDec 2022, 10(12) .
Background: The dismal clinical outcome of relapsed/refractory (R/R) T cell acute lymphoblastic leukemia (T-ALL) highlights the need for innovative targeted therapies. Although chimeric antigen receptor (CAR)-engineered T cells have revolutionized the treatment of B cell malignancies, their clinical implementation in T-ALL is in its infancy. CD1a represents a safe target for cortical T-ALL (coT-ALL) patients, and fratricide-resistant CD1a-directed CAR T cells have been preclinically validated as an immunotherapeutic strategy for R/R coT-ALL. Nonetheless, T-ALL relapses are commonly very aggressive and hyperleukocytic, posing a challenge to recover sufficient non-leukemic effector T cells from leukapheresis in R/R T-ALL patients.
Methods: We carried out a comprehensive study using robust in vitro and in vivo assays comparing the efficacy of engineered T cells either expressing a second-generation CD1a-CAR or secreting CD1a x CD3 T cell-engaging Antibodies (CD1a-STAb).
Results: We show that CD1a-T cell engagers bind to cell surface expressed CD1a and CD3 and induce specific T cell activation. Recruitment of bystander T cells endows CD1a-STAbs with an enhanced in vitro cytotoxicity than CD1a-CAR T cells at lower effector:target ratios. CD1a-STAb T cells are as effective as CD1a-CAR T cells in cutting-edge in vivo T-ALL patient-derived xenograft models.
Conclusions: Our data suggest that CD1a-STAb T cells could be an alternative to CD1a-CAR T cells in coT-ALL patients with aggressive and hyperleukocytic relapses with limited numbers of non-leukemic effector T cells.
Sánchez Martínez D, Tirado N, Mensurado S, Martínez-Moreno A, Romecín P, Gutiérrez Agüera F, Correia DV, Silva-Santos B, Menéndez P
Generation and proof-of-concept for allogeneic CD123 CAR-Delta One T (DOT) cells in acute myeloid leukemia.
Journal for Immunotherapy of Cancer26 Aug 2022, 10(9) .
Background: Chimeric antigen receptor (CAR)-T cells have emerged as a breakthrough treatment for relapse/refractory hematological tumors, showing impressive complete remission rates. However, around 50% of the patients relapse before 1-year post-treatment. T-cell 'fitness' is critical to prolong CAR-T persistence and activity. Allogeneic T cells from healthy donors are less dysfunctional or exhausted than autologous patient-derived T cells; in this context, Delta One T cells (DOTs), a recently described cellular product based on MHC/HLA-independent Vδ1+γδ T cells, represent a promising allogeneic platform.
Methods: Here we generated and preclinically validated, for the first time, 4-1BB-based CAR-DOTs directed against the interleukin-3α chain receptor (CD123), a target antigen widely expressed on acute myeloid leukemia (AML) blasts.
Results: CD123CAR-DOTs showed vigorous, superior to control DOTs, cytotoxicity against AML cell lines and primary samples both in vitro and in vivo, even on tumor rechallenge.
Conclusions: Our results provide the proof-of-concept for a DOT-based next-generation allogeneic CAR-T therapy for AML.
Ramos-Muntada M, Trincado JL, Blanco J, Bueno C, Rodríguez-Cortez VC, Bataller A, López-Millán B, Schwab C, Ortega M, Velasco P, Blanco ML, Nomdedeu J, Ramírez-Orellana M, Minguela A, Fuster JL, Cuatrecasas E, Camós M, Ballerini P, Escherich G, Boer J, denBoer M, Hernández-Rivas JM, Calasanz MJ, Cazzaniga G, Harrison CJ, Menéndez P, Molina O
Clonal heterogeneity and rates of specific chromosome gains are risk predictors in childhood high-hyperdiploid B-cell acute lymphoblastic leukemia
Molecular Oncology21 Jun 2022, . Epub 21 Jun 2022
B-cell acute lymphoblastic leukemia (B-ALL) is the commonest childhood cancer. High hyperdiploidy (HHD) identifies the most frequent cytogenetic subgroup in childhood B-ALL. Although hyperdiploidy represents an important prognostic factor in childhood B-ALL, the specific chromosome gains with prognostic value in HHD-B-ALL remain controversial, and the current knowledge about the hierarchy of chromosome gains, clonal heterogeneity and chromosomal instability in HHD-B-ALL remains very limited. We applied automated sequential-iFISH coupled with single-cell computational modeling to identify the specific chromosomal gains of the eight typically gained chromosomes in a large cohort of 72 primary diagnostic (DX, n=62) and matched relapse (REL, n=10) samples from HHD-B-ALL patients with either favorable or unfavorable clinical outcome in order to characterize the clonal heterogeneity, specific chromosome gains and clonal evolution. Our data show a high degree of clonal heterogeneity and a hierarchical order of chromosome gains in DX samples of HHD-B-ALL. The rates of specific chromosome gains and clonal heterogeneity found in DX samples differ between HHD-B-ALL patients with favorable or unfavorable clinical outcome. In fact, our comprehensive analyses at DX using a computationally-defined risk predictor revealed low levels of trisomies +18+10 and low levels of clonal heterogeneity as robust relapse risk factors in minimal residual disease (MRD)-negative childhood HHD-B-ALL patients: relapse-free survival beyond 5-years: 22.1% vs 87.9%, P<0.0001 and 33.3% vs 80%, P<0.0001, respectively. Moreover, longitudinal analysis of matched DX-REL HHD-B-ALL samples revealed distinct patterns of clonal evolution at relapse. Our study offers a reliable prognostic sub-stratification of pediatric MRD-negative HHD-B-ALL patients.
Zhang YW, Mess J, Aizarani N, Mishra P, Johnson C, Romero-Mulero MC, Rettkowski J, Schönberger K, Obier N, Jäcklein K, Woessner NM, Lalioti ME, Velasco-Hernandez T, Sikora K, Wäsch R, Lehnertz B, Sauvageau G, Manke T, Menendez P, Walter SG, Minguet S, Laurenti E, Günther S, Grün D, Cabezas-Wallscheid N
Hyaluronic acid-GPRC5C signalling promotes dormancy in haematopoietic stem cells.
Nat Cell Biol20 Jun 2022, . Epub 20 Jun 2022
Bone marrow haematopoietic stem cells (HSCs) are vital for lifelong maintenance of healthy haematopoiesis. In inbred mice housed in gnotobiotic facilities, the top of the haematopoietic hierarchy is occupied by dormant HSCs, which reversibly exit quiescence during stress. Whether HSC dormancy exists in humans remains debatable. Here, using single-cell RNA sequencing, we show a continuous landscape of highly purified human bone marrow HSCs displaying varying degrees of dormancy. We identify the orphan receptor GPRC5C, which enriches for dormant human HSCs. GPRC5C is also essential for HSC function, as demonstrated by genetic loss- and gain-of-function analyses. Through structural modelling and biochemical assays, we show that hyaluronic acid, a bone marrow extracellular matrix component, preserves dormancy through GPRC5C. We identify the hyaluronic acid-GPRC5C signalling axis controlling the state of dormancy in mouse and human HSCs.
Blanco B, Ramírez-Fernández Á, Bueno C, Argemí-Muntadas L, Fuentes P, Aguilar-Sopeña Ó, Gutierrez-Agüera F, Zanetti SR, Tapia-Galisteo A, Díez-Alonso L, Segura-Tudela A, Castellà M, Marzal B, Betriu S, Harwood SL, Compte M, Lykkemark S, Erce-Llamazares A, Rubio-Pérez L, Jiménez-Reinoso A, Domínguez-Alonso C, Neves M, Morales P, Paz-Artal E, Guedan S, Sanz L, Toribio ML, Roda-Navarro P, Juan M, Menéndez P, Álvarez-Vallina L
Overcoming CAR-Mediated CD19 Downmodulation and Leukemia Relapse with T Lymphocytes Secreting Anti-CD19 T-cell Engagers.
Cancer Immunol Res1 Apr 2022, 10(4)498-511.
Chimeric antigen receptor (CAR)-modified T cells have revolutionized the treatment of CD19-positive hematologic malignancies. Although anti-CD19 CAR-engineered autologous T cells can induce remission in patients with B-cell acute lymphoblastic leukemia, a large subset relapse, most of them with CD19-positive disease. Therefore, new therapeutic strategies are clearly needed. Here, we report a comprehensive study comparing engineered T cells either expressing a second-generation anti-CD19 CAR (CAR-T19) or secreting a CD19/CD3-targeting bispecific T-cell engager antibody (STAb-T19). We found that STAb-T19 cells are more effective than CAR-T19 cells at inducing cytotoxicity, avoiding leukemia escape in vitro, and preventing relapse in vivo. We observed that leukemia escape in vitro is associated with rapid and drastic CAR-induced internalization of CD19 that is coupled with lysosome-mediated degradation, leading to the emergence of transiently CD19-negative leukemic cells that evade the immune response of engineered CAR-T19 cells. In contrast, engineered STAb-T19 cells induce the formation of canonical immunologic synapses and prevent the CD19 downmodulation observed in anti-CD19 CAR-mediated interactions. Although both strategies show similar efficacy in short-term mouse models, there is a significant difference in a long-term patient-derived xenograft mouse model, where STAb-T19 cells efficiently eradicated leukemia cells, but leukemia relapsed after CAR-T19 therapy. Our findings suggest that the absence of CD19 downmodulation in the STAb-T19 strategy, coupled with the continued antibody secretion, allows an efficient recruitment of the endogenous T-cell pool, resulting in fast and effective elimination of cancer cells that may prevent CD19-positive relapses frequently associated with CAR-T19 therapies.
Lopez-Millan B, Costales P, Gutiérrez-Agüera F, Díaz de la Guardia R, Roca-Ho H, Vinyoles M, Rubio-Gayarre A, Safi R, Castaño J, Romecín PA, Ramírez-Orellana M, Anguita E, Jeremias I, Zamora L, Rodríguez-Manzaneque JC, Bueno C, Morís F, Menendez P
The Multi-Kinase Inhibitor EC-70124 Is a Promising Candidate for the Treatment of FLT3-ITD-Positive Acute Myeloid Leukemia.
Cancers (Basel)21 Mar 2022, 14(6) . Epub 21 Mar 2022
Acute myeloid leukemia (AML) is the most common acute leukemia in adults. Patients with AML harboring a constitutively active internal tandem duplication mutation (ITDMUT) in the FMS-like kinase tyrosine kinase (FLT3) receptor generally have a poor prognosis. Several tyrosine kinase/FLT3 inhibitors have been developed and tested clinically, but very few (midostaurin and gilteritinib) have thus far been FDA/EMA-approved for patients with newly diagnosed or relapse/refractory FLT3-ITDMUT AML. Disappointingly, clinical responses are commonly partial or not durable, highlighting the need for new molecules targeting FLT3-ITDMUT AML. Here, we tested EC-70124, a hybrid indolocarbazole analog from the same chemical space as midostaurin with a potent and selective inhibitory effect on FLT3. In vitro, EC-70124 exerted a robust and specific antileukemia activity against FLT3-ITDMUT AML primary cells and cell lines with respect to cytotoxicity, CFU capacity, apoptosis and cell cycle while sparing healthy hematopoietic (stem/progenitor) cells. We also analyzed its efficacy in vivo as monotherapy using two different xenograft models: an aggressive and systemic model based on MOLM-13 cells and a patient-derived xenograft model. Orally disposable EC-70124 exerted a potent inhibitory effect on the growth of FLT3-ITDMUT AML cells, delaying disease progression and debulking the leukemia. Collectively, our findings show that EC-70124 is a promising and safe agent for the treatment of AML with FLT3-ITDMUT.
Oscar Molina, Alex Bataller, Namitha Thampi, Jordi Ribera, Isabel Granada, Pablo Velasco, José Luis Fuster, Pablo Menéndez
Near-Haploidy and Low-Hypodiploidy in B-Cell Acute Lymphoblastic Leukemia: When Less Is Too Much
Cancers 2022, 14(1), 32; https://doi.org/10.3390/cancers1401003222 Dec 2021, .
Hypodiploidy with less than 40 chromosomes is a rare genetic abnormality in B-cell acute lymphoblastic leukemia (B-ALL). This condition can be classified based on modal chromosome number as low-hypodiploidy (30–39 chromosomes) and near-haploidy (24–29 chromosomes), with unique cytogenetic and mutational landscapes. Hypodiploid B-ALL with <40 chromosomes has an extremely poor outcome, with 5-year overall survival rates below 50% and 20% in childhood and adult B-ALL, respectively. Accordingly, this genetic feature represents an adverse prognostic factor in B-ALL and is associated with early relapse and therapy refractoriness. Notably, half of all patients with hypodiploid B-ALL with < 40 chromosomes cases ultimately exhibit chromosome doubling of the hypodiploid clone, resulting in clones with 50–78 chromosomes. Doubled clones are often the major clones at diagnosis, leading to “masked hypodiploidy”, which is clinically challenging as patients can be erroneously classified as hyperdiploid B-ALL. Here, we summarize the main cytogenetic and molecular features of hypodiploid B-ALL subtypes, and provide a brief overview of the diagnostic methods, standard-of-care treatments and overall clinical outcome. Finally, we discuss molecular mechanisms that may underlie the origin and leukemogenic impact of hypodiploidy and may open new therapeutic avenues to improve survival rates in these patients.
Ribeiro ML, Reyes-Garau D, Vinyoles M, Profitos-Peleja N, Santos JC, Armengol M, Fernández-Serrano M, Sedo Mor A, Bech-Serra JJ, Blecua P, Musulen E, De La Torre C, Miskin HP, Esteller M, Bosch F, Menéndez P, Normant E, Roué G
Antitumor activity of the novel BTK inhibitor TG-1701 is associated with disruption of Ikaros signaling in patients with B-cell non-Hodgkin lymphoma.
Clin Cancer Res2 Dec 2021, . Epub 22 Sep 2021
Purpose: Despite the remarkable activity of BTK inhibitors (BTKi) in relapsed B-cell non-Hodgkin lymphoma (B-NHL), no clinically-relevant biomarker has been associated to these agents so far. The relevance of phosphoproteomic profiling for the early identification of BTKi responders remains underexplored.
Experimental design: A set of six clinical samples from an ongoing phase 1 trial dosing chronic lymphocytic leukemia (CLL) patients with TG-1701, a novel irreversible and highly specific BTKi, were characterized by phosphoproteomic and RNA-seq analysis. The activity of TG-1701 was evaluated in a panel of eleven B-NHL cell lines and mouse xenografts, including two NFκB- and BTKC481S-driven BTKi resistant models. Biomarker validation and signal transduction analysis were conducted through real-time PCR, western blot, immunostaining and gene knock-out (KO) experiments.
Results: A non-supervised, phosphoproteomic-based clustering did match the early clinical outcomes of CLL patients and separated a group of "early-responders" from a group of "late-responders". This clustering was based on a selected list of 96 phosphosites with Ikaros-pSer442/445 as a potential biomarker for TG-1701 efficacy. TG-1701 treatment was further shown to blunt Ikaros gene signature, including YES1 and MYC, in early-responder patients as well as in BTKi-sensitive B-NHL cell lines and xenografts. In contrast, Ikaros nuclear activity and signaling remained unaffected by the drug in vitro and in vivo, in late-responder patients and in BTKC481S, BTKKO and non-canonical NFκB models.
Conclusions: These data validate phosphoproteomic as a valuable tool for the early detection of response to BTK inhibition in the clinic, and for the determination of drug mechanism of action.