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

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

In vitro assessment of the role of p53 on chemotherapy treatments in neuroblastoma cell lines

Neuroblastoma is the most frequent malignant extracranial solid tumor of infancy. The overall objective of this work consists of determining the presence of alterations in the p53/MDM2/p14ARF signaling pathway in neuroblastoma cell lines and deciphering their possible relationship with resistance to known antineoplastic drugs and to differentiation agents. Firstly, we characterized 10 neuroblastoma cell lines for alterations at the p53/MDM2/p14ARF signaling pathway by analysis of TP53 point mutations, MYCN and MDM2 amplification, and p14ARF methylation, homozygous deletions, and expression. Secondly, we chose SK-N-FI (mutated at TP53) and SK-N-Be(2) (wild-type TP53) cell lines, treated them with chemotherapeutic agents (doxorubicin, etoposide, cisplatin, and melphalan) and with two isomers of retinoic acid (RA): (9-cis and all-trans). Finally, we analyzed the distribution of the cell cycle, the induction of apoptosis, and the expression levels of p53, p21, and Bcl-2 in those two cell lines. P14ARF did not present promoter methylation, homozygous deletions, and protein expression in any of the 10 neuroblastoma cell lines. One TP53 point mutation was detected in the SK-N-FI cell line. MYCN amplification was frequent, while most cell lines did not present MDM2 amplification. Treatment of SK-N-FI and SK-N-Be(2) cells with doxorubicin, etoposide, cisplatin, and melphalan increased apoptosis and blocked the cycle in G2/M, while retinoic acid isomers induced apoptosis and decreased the percentage of cells in S phase in TP53 mutated SK-N-FI cells, but not in TP53 wild-type SK-N-Be(2) cells. Treatment with cisplatin, melphalan, or 9-cis RA decreased p53 expression levels in SK-N-FI cells but not in SK-N-Be (2). The expression of p21 was not modified in either of the two cell lines. Bcl-2 levels were reduced only in SK-N-FI cells after treatment with cisplatin. However, treatments with doxorubicin, etoposide, or 9-cis-RA did not modify the levels of this protein in either of the two cell lines. In conclusion, TP53 mutated SK-N-FI cells respond better to the retinoic isomers than TP53 wild-type SK-N-Be(2) cells. Although these are in vitro results, it seems that deciphering the molecular alterations of the p53/MDM2/p14ARF signaling pathway prior to treating patients of neuroblastoma might be useful for standardizing therapies with the aim of improving survival

In vitro assessment of the role of p53 on chemotherapy treatments in neuroblastoma cell lines

Neuroblastoma is the most frequent malignant extracranial solid tumor of infancy. The overall objective of this work consists of determining the presence of alterations in the p53/MDM2/p14ARF signaling pathway in neuroblastoma cell lines and deciphering their possible relationship with resistance to known antineoplastic drugs and to differentiation agents. Firstly, we characterized 10 neuroblastoma cell lines for alterations at the p53/MDM2/p14ARF signaling pathway by analysis of TP53 point mutations, MYCN and MDM2 amplification, and p14ARF methylation, homozygous deletions, and expression. Secondly, we chose SK-N-FI (mutated at TP53) and SK-N-Be(2) (wild-type TP53) cell lines, treated them with chemotherapeutic agents (doxorubicin, etoposide, cisplatin, and melphalan) and with two isomers of retinoic acid (RA): (9-cis and all-trans). Finally, we analyzed the distribution of the cell cycle, the induction of apoptosis, and the expression levels of p53, p21, and Bcl-2 in those two cell lines. P14ARF did not present promoter methylation, homozygous deletions, and protein expression in any of the 10 neuroblastoma cell lines. One TP53 point mutation was detected in the SK-N-FI cell line. MYCN amplification was frequent, while most cell lines did not present MDM2 amplification. Treatment of SK-N-FI and SK-N-Be(2) cells with doxorubicin, etoposide, cisplatin, and melphalan increased apoptosis and blocked the cycle in G2/M, while retinoic acid isomers induced apoptosis and decreased the percentage of cells in S phase in TP53 mutated SK-N-FI cells, but not in TP53 wild-type SK-N-Be(2) cells. Treatment with cisplatin, melphalan, or 9-cis RA decreased p53 expression levels in SK-N-FI cells but not in SK-N-Be (2). The expression of p21 was not modified in either of the two cell lines. Bcl-2 levels were reduced only in SK-N-FI cells after treatment with cisplatin. However, treatments with doxorubicin, etoposide, or 9-cis-RA did not modify the levels of this protein in either of the two cell lines. In conclusion, TP53 mutated SK-N-FI cells respond better to the retinoic isomers than TP53 wild-type SK-N-Be(2) cells. Although these are in vitro results, it seems that deciphering the molecular alterations of the p53/MDM2/p14ARF signaling pathway prior to treating patients of neuroblastoma might be useful for standardizing therapies with the aim of improving survival

Chronic docosahexaenoic acid supplementation improves metabolic plasticity in subcutaneous adipose tissue of aged obese female mice

This study aimed to characterize the potential beneficial effects of chronic docosahexaenoic acid (DHA) supplementation on restoring subcutaneous white adipose tissue (scWAT) plasticity in obese aged female mice. Two-month-old female C57BL/6J mice received a control (CT) or a high fat diet (HFD) for 4 months. Then, 6-month-old diet-induced obese (DIO) mice were distributed into the DIO and the DIOMEG group (fed with a DHA-enriched HFD) up to 18 months. In scWAT, the DHA-enriched diet reduced the mean adipocyte size and reversed the upregulation of lipogenic genes induced by the HFD, reaching values even lower than those observed in CT animals. DIO mice exhibited an up-regulation of lipolytic and fatty oxidation gene expressions that was reversed in DHA-supplemented mice except for Cpt1a mRNA levels, which were higher in DIOMEG as compared to CT mice. DHA restored the increase of proinflammatory genes observed in scWAT of DIO mice. While no changes were observed in total macrophage F4/80+/CD11b+ content, the DHA treatment switched scWAT macrophages profile by reducing the M1 marker Cd11c and increasing the M2 marker CD206. These events occurred alongside with a stimulation of beige adipocyte specific genes, the restoration of UCP1 and pAKT/AKT ratio, and a recovery of the HFD-induced Fgf21 upregulation. In summary, DHA supplementation induced a metabolic remodeling of scWAT to a healthier phenotype in aged obese mice by modulating genes controlling lipid accumulation in adipocytes, reducing the inflammatory status, and inducing beige adipocyte markers in obese aged mice

Chronic docosahexaenoic acid supplementation improves metabolic plasticity in subcutaneous adipose tissue of aged obese female mice

This study aimed to characterize the potential beneficial effects of chronic docosahexaenoic acid (DHA) supplementation on restoring subcutaneous white adipose tissue (scWAT) plasticity in obese aged female mice. Two-month-old female C57BL/6J mice received a control (CT) or a high fat diet (HFD) for 4 months. Then, 6-month-old diet-induced obese (DIO) mice were distributed into the DIO and the DIOMEG group (fed with a DHA-enriched HFD) up to 18 months. In scWAT, the DHA-enriched diet reduced the mean adipocyte size and reversed the upregulation of lipogenic genes induced by the HFD, reaching values even lower than those observed in CT animals. DIO mice exhibited an up-regulation of lipolytic and fatty oxidation gene expressions that was reversed in DHA-supplemented mice except for Cpt1a mRNA levels, which were higher in DIOMEG as compared to CT mice. DHA restored the increase of proinflammatory genes observed in scWAT of DIO mice. While no changes were observed in total macrophage F4/80+/CD11b+ content, the DHA treatment switched scWAT macrophages profile by reducing the M1 marker Cd11c and increasing the M2 marker CD206. These events occurred alongside with a stimulation of beige adipocyte specific genes, the restoration of UCP1 and pAKT/AKT ratio, and a recovery of the HFD-induced Fgf21 upregulation. In summary, DHA supplementation induced a metabolic remodeling of scWAT to a healthier phenotype in aged obese mice by modulating genes controlling lipid accumulation in adipocytes, reducing the inflammatory status, and inducing beige adipocyte markers in obese aged mice

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

Richter transformation driven by Epstein-Barr virus reactivation during therapy-related immunosuppression in chronic lymphocytic leukemia

The increased risk of Richter transformation (RT) in patients with chronic lymphocytic leukemia (CLL) due to Epstein-Barr virus (EBV) reactivation during immunosuppressive therapy with fludarabine or other targeted agents remains controversial. Among 31 RT cases classified as diffuse large B-cell lymphoma (DLBCL), seven (23%) showed EBV expression. In comparison to EBV-negative tumors, EBV+ DLBCLs derived predominantly from IGVH-hypermutated CLL and exhibited CLL-unrelated IGVH sequences more frequently. Intriguingly, despite having different cellular origins, clonally related and unrelated EBV+ DLBCLs shared a previous history of immunosuppressive chemo-immunotherapy, a non-germinal center DLBCL phenotype, EBV latency programs type II/III, and a very short survival. These data suggested that EBV reactivation during therapy-related immunosuppression can transform either CLL cells or non-tumoral B lymphocytes into EBV+ DLBCL. To investigate this hypothesis, xenogeneic transplantation of blood cells from 31 patients with CLL and monoclonal B-cell lymphocytosis (MBL) was performed in Rag2-/-IL2γc-/-mice. Remarkably, the recipients' impaired immunosurveillance favored the spontaneous outgrowth of EBV+ B-cell clones from 95% of CLL and 64% of MBL patients, but not from healthy donors. Eventually, these cells generated monoclonal tumors (mostly CLL-unrelated but also CLL-related) recapitulating the principal features of EBV+ DLBCL in patients. Accordingly, clonally related and unrelated EBV+ DLBCL xenografts exhibited indistinguishable cellular, virological and molecular features, and synergistically responded to combined inhibition of EBV replication with ganciclovir and BCR signaling with ibrutinib in vivo. Our study underscores the risk of RT driven by EBV in CLL patients receiving immunosuppressive therapies, and provides the scientific rationale for testing ganciclovir and ibrutinib in EBV+ DLBCL