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

Changes in gene expression profiling of apoptotic genes in neuroblastoma cell lines upon retinoic acid treatment.

To determine the effect of retinoic acid (RA) in neuroblastoma we treated RA sensitive neuroblastoma cell lines with 9-cis RA or ATRA for 9 days, or for 5 days followed by absence of RA for another 4 days. Both isomers induced apoptosis and reduced cell density as a result of cell differentiation and/or apoptosis. Flow cytometry revealed that 9-cis RA induced apoptosis more effectively than ATRA. The expression profile of apoptosis and survival pathways was cell line specific and depended on the isomer used

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

Effect of RA treatment over apoptotic and cell survival pathways in SK-N-DZ RA sensitive cell line.

<p>In green colour are represented those genes whose expression has been augmented as a result of 9-<i>cis</i> RA (A) or ATRA (B) treatment, while in red are shown those whose expression has been decreased. Green arrows indicate the pathways that will be activated while red arrows indicate the pathways that will be inhibited as a consequence of the modifications of the genes that take part in these pathways. CM: Cell Membrane; Cyt: Cytoplasm; Mit: Mitochondrion; Nuc: Nucleus; P: Phosphorous.</p

Genes of the apoptotic pathway overexpressed at least two times, or downregulated by 50%, after treatment of neuroblastoma cell lines with ATRA.

<p>Genes significantly upregulated or downregulated more than 2-fold are represented. Asterisks indicate significant differences among control and treated cells (*, p<0.05; **, p<0.01). Lines (–) indicate that these genes were neither overexpressed at least two times, nor downregulated by 50% after the treatment.</p

Time course analysis of the differentiation process induced in neuroblastoma cell cultures by Retinoic Acid.

<p>Expression levels of the oncogenes RET and MYCN were measured in IMR-32, SH-SY5Y, SK-N-DZ, SK-N-Be(2) and SK-N-AS cell cultures either untreated (control cells) or treated with 9<i>-cis</i> RA or ATRA for 9 consecutive days (RA(9)), or treated for 5 days followed by 4 days in the absence of RA (RA(5)). Expression levels were measured by qPCR as described in Materials and Methods and are shown relative to GAPDH. RET levels in cells either untreated (control) or treated with (A) 9-<i>cis</i> RA or (B) ATRA were determined at days 1, 3, 5, 7 and 9. MYCN levels in cells either untreated (control) or treated with (C) 9<i>-cis</i> RA or (D) ATRA were measured at day 9. Data are represented as mean ± SEM (n = 3). Asterisks represent significant differences to the control (*, p<0.05; **, p<0.01).</p

Effect of RA treatment over apoptotic and cell survival pathways in SK-N-Be(2) RA sensitive cell line.

<p>In green colour are represented those genes whose expression has been augmented as a result of 9-<i>cis</i> RA (A) or ATRA (B) treatment, while in red are shown those whose expression has been decreased. Green arrows indicate the pathways that will be activated while red arrows indicate the pathways that will be inhibited as a consequence of the modifications of the genes that take part in these pathways. CM: Cell Membrane; Cyt: Cytoplasm; Mit: Mitochondrion; Nuc: Nucleus; P: Phosphorous.</p

Time course analysis of induction of apoptosis in neuroblastoma cell cultures by Retinoic Acid.

<p>Percentage of dead cells in IMR-32, SH-SY5Y, SK-N-DZ, SK-N-Be(2) and SK-N-AS cell cultures either untreated (control cells) or treated with (A) 9-<i>cis</i> RA or (B) ATRA for 9 consecutive days (RA(9)) or for 5 days followed by 4 days in the absence of RA (RA(5)) was determined by TUNEL after 1, 3, 5, 7 or 9 days of treatment. Data are represented as mean ± SEM (n = 3). Asterisks represent significant differences to the control (*, p<0.05; **, p<0.01).</p