Discovery of first-in-class reversible dual small molecule inhibitors against G9a and DNMTs in hematological malignancies

The indisputable role of epigenetics in cancer and the fact that epigenetic alterations can be reversed have favoured development of epigenetic drugs. In this study, we design and synthesize potent novel, selective and reversible chemical probes that simultaneously inhibit the G9a and DNMTs methyltransferase activity. In vitro treatment of haematological neoplasia (acute myeloid leukaemia-AML, acute lymphoblastic leukaemia-ALL and diffuse large B-cell lymphoma-DLBCL) with the lead compound CM-272, inhibits cell proliferation and promotes apoptosis, inducing interferon-stimulated genes and immunogenic cell death. CM-272 significantly prolongs survival of AML, ALL and DLBCL xenogeneic models. Our results represent the discovery of first-in-class dual inhibitors of G9a/DNMTs and establish this chemical series as a promising therapeutic tool for unmet needs in haematological tumours.We particularly acknowledge the Biobank of the University of Navarra for its collaboration. We thank Dr Edorta Martínez de Marigorta and Dr Francisco Palacios from Departamento de Química Orgánica I, Facultad de Farmacia, Universidad del Pais Vasco for 13C NMR determination and Angel Irigoyen Barrio and Dr Ana Romo Hualde, from University of Navarra, for HRMS determination. Dr. Irene de Miguel Turrullols from Small Molecule Discovery Platform, CIMA, University of Navarra is acknowledged for NMR data interpretation. This work was funded by grants from Instituto de Salud Carlos III (ISCIII) PI10/01691, PI13/01469, PI14/01867, PI10/2983, TRASCAN (EPICA), CIBERONC, cofinanciacion FEDER, RTICC RD12/0036/0068, Fundació La Marató de TV3 (20132130-31-32) and ‘Fundación Fuentes Dutor’. B.P. is supported by a Sara Borrell fellowship CD13/00340 and X.A. is a Marie Curie researcher under contract ‘LincMHeM-330598’.S

Preclinical models for prediction of immunotherapy outcomes and immune evasion mechanisms in genetically heterogeneous multiple myeloma

The historical lack of preclinical models reflecting the genetic heterogeneity of multiple myeloma (MM) hampers the advance of therapeutic discoveries. To circumvent this limitation, we screened mice engineered to carry eight MM lesions (NF-κB, KRAS, MYC, TP53, BCL2, cyclin D1, MMSET/NSD2 and c-MAF) combinatorially activated in B lymphocytes following T cell-driven immunization. Fifteen genetically diverse models developed bone marrow (BM) tumors fulfilling MM pathogenesis. Integrative analyses of ∼500 mice and ∼1,000 patients revealed a common MAPK-MYC genetic pathway that accelerated time to progression from precursor states across genetically heterogeneous MM. MYC-dependent time to progression conditioned immune evasion mechanisms that remodeled the BM microenvironment differently. Rapid MYC-driven progressors exhibited a high number of activated/exhausted CD8+ T cells with reduced immunosuppressive regulatory T (Treg) cells, while late MYC acquisition in slow progressors was associated with lower CD8+ T cell infiltration and more abundant Treg cells. Single-cell transcriptomics and functional assays defined a high ratio of CD8+ T cells versus Treg cells as a predictor of response to immune checkpoint blockade (ICB). In clinical series, high CD8+ T/Treg cell ratios underlie early progression in untreated smoldering MM, and correlated with early relapse in newly diagnosed patients with MM under Len/Dex therapy. In ICB-refractory MM models, increasing CD8+ T cell cytotoxicity or depleting Treg cells reversed immunotherapy resistance and yielded prolonged MM control. Our experimental models enable the correlation of MM genetic and immunological traits with preclinical therapy responses, which may inform the next-generation immunotherapy trials

Prolactin-induced mouse mammary carcinomas model estrogen resistant luminal breast cancer.

INTRODUCTION: Tumors that express estrogen receptor alpha (ERα+) comprise 75% of breast cancers in women. While treatments directed against this receptor have successfully lowered mortality rates, many primary tumors initially or later exhibit resistance. The paucity of murine models of this luminal tumor subtype has hindered studies of factors that promote their pathogenesis and modulate responsiveness to estrogen-directed therapeutics. Since epidemiologic studies closely link prolactin and the development of ERα+ tumors in women, we examined characteristics of the aggressive ERα+ and ERα- carcinomas which develop in response to mammary prolactin in a murine transgenic model (neu-related lipocalin- prolactin (NRL-PRL)). To evaluate their relationship to clinical tumors, we determined phenotypic relationships among these carcinomas, other murine models of breast cancer, and features of luminal tumors in women. METHODS: We examined a panel of prolactin-induced tumors for characteristics relevant to clinical tumors: histotype, ERα/progesterone receptor (PR) expression and estrogen responsiveness, Activating Protein 1 (AP-1) components, and phosphorylation of signal transducer and activator of transcription 5 (Stat5), extracellular signal regulated kinase (ERK) 1/2 and AKT. We compared levels of transcripts in the ERα-associated luminal signature that defines this subtype of tumors in women and transcripts enriched in various mammary epithelial lineages to other well-studied genetically modified murine models of breast cancer. Finally, we used microarray analyses to compare prolactin-induced ERα+ and ERα- tumors, and examined responsiveness to estrogen and the anti-estrogen, Faslodex, in vivo. RESULTS: Prolactin-induced carcinomas were markedly diverse with respect to histotype, ERα/PR expression, and activated signaling cascades. They constituted a heterogeneous, but distinct group of murine mammary tumors, with molecular features of the luminal subtype of human breast cancer. In contrast to morphologically normal and hyperplastic structures in NRL-PRL females, carcinomas were insensitive to ERα-mediated signals. These tumors were distinct from mouse mammary tumor virus (MMTV)-neu tumors, and contained elevated transcripts for factors associated with luminal/alveolar expansion and differentiation, suggesting that they arose from physiologic targets of prolactin. These features were shared by ERα+ and ERα- tumors, suggesting a common origin, although the former exhibited transcript profiles reflecting greater differentiation. CONCLUSIONS: Our studies demonstrate that prolactin can promote diverse carcinomas in mice, many of which resemble luminal breast cancers, providing a novel experimental model to examine the pathogenesis, progression and treatment responsiveness of this tumor subtype

Preneoplastic somatic mutations including MYD88(L265P) in lymphoplasmacytic lymphoma

Normal cell counterparts of solid and myeloid tumors accumulate mutations years before disease onset; whether this occurs in B lymphocytes before lymphoma remains uncertain. We sequenced multiple stages of the B lineage in elderly individuals and patients with lymphoplasmacytic lymphoma, a singular disease for studying lymphomagenesis because of the high prevalence of mutated MYD88. We observed similar accumulation of random mutations in B lineages from both cohorts and unexpectedly found MYD88(L265P) in normal precursor and mature B lymphocytes from patients with lymphoma. We uncovered genetic and transcriptional pathways driving malignant transformation and leveraged these to model lymphoplasmacytic lymphoma in mice, based on mutated MYD88 in B cell precursors and BCL2 overexpression. Thus, MYD88(L265P) is a preneoplastic event, which challenges the current understanding of lymphomagenesis and may have implications for early detection of B cell lymphomas

PD-1/PD-L1 immune checkpoint and p53 loss facilitate tumor progression in activated B-cell diffuse large B-cell lymphomas

Refractory or relapsed diffuse large B-cell lymphoma (DLBCL) often associates with the activated B-cell-like (ABC) subtype and genetic alterations that drive constitutive NF-κB activation and impair B-cell terminal differentiation. Here, we show that DNA damage response by p53 is a central mechanism suppressing the pathogenic cooperation of IKK2ca-enforced canonical NF-κB and impaired differentiation resulting from Blimp1 loss in ABC-DLBCL lymphomagenesis. We provide evidences that the interplay between these genetic alterations and the tumor microenvironment select for additional molecular addictions that promote lymphoma progression, including aberrant coexpression of FOXP1 and the B-cell mutagenic enzyme activation-induced deaminase, and immune evasion through major histocompatibility complex class II downregulation, PD-L1 upregulation, and T-cell exhaustion. Consistently, PD-1 blockade cooperated with anti-CD20-mediated B-cell cytotoxicity, promoting extended T-cell reactivation and antitumor specificity that improved long-term overall survival in mice. Our data support a pathogenic cooperation among NF-κB-driven prosurvival, genetic instability, and immune evasion mechanisms in DLBCL and provide preclinical proof of concept for including PD-1/PD-L1 blockade in combinatorial immunotherapy for ABC-DLBCL.status: publishe

Preneoplastic somatic mutations including MYD88L265P in lymphoplasmacytic lymphoma

: Normal cell counterparts of solid and myeloid tumors accumulate mutations years before disease onset; whether this occurs in B lymphocytes before lymphoma remains uncertain. We sequenced multiple stages of the B lineage in elderly individuals and patients with lymphoplasmacytic lymphoma, a singular disease for studying lymphomagenesis because of the high prevalence of mutated MYD88. We observed similar accumulation of random mutations in B lineages from both cohorts and unexpectedly found MYD88L265P in normal precursor and mature B lymphocytes from patients with lymphoma. We uncovered genetic and transcriptional pathways driving malignant transformation and leveraged these to model lymphoplasmacytic lymphoma in mice, based on mutated MYD88 in B cell precursors and BCL2 overexpression. Thus, MYD88L265P is a preneoplastic event, which challenges the current understanding of lymphomagenesis and may have implications for early detection of B cell lymphomas

Remodeling of the Immune Microenvironment By Oncogenic MYD88 Dictates Immunotherapy Responses across Indolent and Aggressive B-Cell Lymphomas

The MYD88 L265P mutation is detected in a variety of lymphoma subtypes, ranging from indolent MALT or lymphoplasmacytic lymphoma (LPL) to aggressive DLBCL with activated B-cell (ABC) phenotype. Whether oncogenic MYD88 L265P constitutes a common founder mutation and how it drives lymphomas with different clinical, histopathological, and immunological characteristics, remains unknown. To address this fundamental question, we screened mice genetically engineered to express an orthologous MYD88 L252P mutation crossed with animals carrying secondary genetic lesions common in MYD88 L265P lymphomas, triggered at two B-cell differentiation stages. Several mouse strains developed indolent or aggressive lymphomas depending on the cell of origin and the secondary genetic lesion. In two of the models, human-like MALT or LPL were progressively driven by mutant MYD88 activated from immature pre-B lymphocytes with either constitutive BCR signaling or BCL2 expression. High-throughput cellular and molecular characterization of the tumor immune microenvironment (TME) by single-cell RNA sequencing coupled with BCR/TCRseq revealed bi-directional B-cell interactions with CD4 + Tfh and Th1 cells from early disease state, promoting survival of B-cell clones through CD40L:CD40 signaling. During progression, IL10-secreting regulatory T (Treg) cells were recruited into the tumor niche through a Ccl5-Cxcr3 axis to suppress CD8 + T-cell cytotoxicity. In patients with MYD88 L265P LPL characterized at progressive disease states, CD40:CD40L interaction between tumor and immune cells took place early while Tregs accumulated at late stage. In the LPL model and in human MYD88 L265P-LPL cells, CD40 signaling decreased effects of standard-of-care drugs including BTK inhibitors. Accordingly, anti-CD40 therapy disrupted B/T-cell interactions and increased ibrutinib activity. In conclusion, oncogenic MYD88 triggered at early B-cell stages promotes indolent lymphomas that are sustained by CD40 signaling, which restricts therapy responses that can be restored upon CD40 blockade. When MYD88 L265P was activated in mature germinal-center B cells together with either Prdm1 or Trp53 deletion, mice developed extranodal ABC-DLBCL (MCD/C5 genomic class). IHC, flow cytometry and single-cell RNAseq revealed that DLBCL with Prdm1 loss showed morphologically heterogeneous B cells with abundant immune and T cells with activated/exhausted phenotypes, which recapitulated the inflammatory/immunosuppressive TME category in DLBCL patients (IN-LME, Kotlov- Cancer-Discovery-2021). In contrast, DLBCL with Tp53 deletion exhibited monotonous B-cell infiltrates with high proliferation index and a TME with depletion of T cells with effector/memory phenotypes and CD4 +CD25 +Foxp3 + Tregs, consistent with the immune depleted TME category (DP-LME). Analysis of 1,039 DLBCL patients demonstrated that majority of MCD cases with MYD88 L265P showed either an IN-LME (43%) or DP-LME (44%). Consistent with the models, ABC-DLBCL patients with MYD88 L265P and TP53 loss/mutation exhibited highly proliferative disease with a DP-LME, while ABC-DLBCLs with MYD88 L265P but without TP53 abnormalities showed an IN-LME. Notably, the number of infiltrating CD4 + T cells correlated with TP53 activity score (PROGeNY) across MCD-DLBCLs. Overall, the presence or absence of TP53 alterations in MYD88 L265P ABC-DLBCL induces TME characterized as DP-LME or IN-LME, respectively. Since DP-LME carries poor response to immune-chemotherapy (Kotlov-Cancer-Discovery-2021), we tested anti-CD19 CAR T-cells in the models. While CAR T-cells were initially effective in the two DLBCL models, progression occurred earlier in DLBCL with DP-LME than in IN-LME, which remained in remission 100 days after cell infusion. Responses correlated with persistence of circulating CD4 + CAR T cells. These data highlight that a TP53-driven DP-LME may limit immunotherapy activity in DLBCL, providing a scientific explanation to the poor outcome of DLBCL patients with TP53 alterations to CD19-directed CAR T cells (Shouval-JCO-2021). Collectively, our study defines the cellular origins and the stepwise genetic alterations of indolent and aggressive lymphomas with MYD88 L265P, dissects distinct immunological paths underlying progression driven by oncogenic MYD88, and builds proof-of-concept for advancing precision immunotherapy in B-cell lymphomas