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was highly expressed in no responsive ABC-DLBCL patients after Ibrutinib treatment.
C481S BTK HBL-1 cells were treated with 0–8 uM Ibrutinib or combination with 17.5 uM FX1.
value of Ibrutinib compared with Ibrutinib combining FX1. Values were calculated mean ± SD (
Discussion
Even though patients of DLBCL respond sensitively to first-line treatment, approximately 40% of patients still could not benefit from it [
4
]. Ibrutinib is an inhibitor of BTK, showed obvious efficacy in rel/ref DLBCL, especially ABC subtype [
11
]. A phase I study of Ibrutinib combined rituximab, ifosfamide, carboplatin, and etoposide (R-ICE) for patients with rel/ref DLBCL reported the high rate of overall response of 90%, including 11 patients achieved complete remission (CR) and 7 patients partial remission (PR) [
19
]. And another phase III study indicated Ibrutinib and rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) improved event-free survival (EFS), progression-free survival (PFS), overall survival (OS) in ABC DLBCL patients [
20
]. Increasing clinical trials investigate Ibrutinib curative effect on DLBCL. We knew that Ibrutinib was approved to be used for several B-cell malignancies in FDA in 2013 [
10
]. However, the emergence of Ibrutinib resistance has limited its efficacy [
21
,
22
], so it’s important to study the mechanism of Ibrutinib resistance in DLBCL.
In this study, three microarray expression profile datasets were analyzed to further study mechanism of Ibrutinib resistance. First, a total of 237 common DEGs between Ibrutinib sensitive and resistant cell lines were identified through two expression profiles, including 100 upregulated and 137 downregulated genes. Second, GO analysis was performed to study functional roles, including biological process, molecular functions and cellular component. The upregulated DEGs were correlated with transcription and downregulated DEGs were signal transduction and negative regulation of apoptotic process. Third, signal pathway enrichment analysis showed that upregulated DEGs were mainly enriched in cell cycle and downregulated DEGs were mainly enriched in cytokines-based pathways. What is more, PPI network was conducted to illustrate interactions among the DEGs at the protein level, of which, a total of 7 genes-
BCL6
,
IL10
,
IL2RB
,
IRF4
,
CD80
,
PMDR1
,
GZMB
- were selected as hub genes with the threshold of MCC score > 10,000. And through predicting miRNA associated with hub genes, we found that miR-30 family may be related to Ibrutinib resistance in ABC-DLBCL. From the cBioportal database, we found that
BCL6
showed the highest level of genetic alterations among above hub genes. Using another expression profile, we found that
BCL6
highly expressed in no responsive ABC-DLBCL patients, and the AUC of ROC curve was 0.67.
BCL6
, a transcription repressor, plays an important role of initiation and maintenance of germinal center reactions [
23
,
24
], which has been identified as one of predictors of outcome in several cancers, such as DLBCL and B-cell acute lymphoblastic leukemia (B-ALL) [
25
,
26
]. It was reported that
BCL6
is associated with tyrosine kinase inhibitors (TKI) resistance in Philadelphia chromosome positive (Ph+) ALL and chronic myeloid leukemia (CML) cells [
27
,
28
]. And another study showed overexpression of
BCL6
inhibited the sensitivity of methotrexate in children with B-ALL by promoting
ZEB1
expression [
29
]. What is more, Julie et al. found association between
BCL6
overexpression and etoposide resistance in DLBCL cell lines [
30
]. Cardenas et al. reported that
BCL6
expresses in most ABC-DLBCL at a low level [
31
]. It was interesting that
BCL6
was upregulated in Ibrutinib-resistant ABC-DLBCL cell lines in our study. And
BCL6
had the highest MCC score in Ibrutinib-resistant PPI network and linked with another hub genes. So using another gene profile validated that
BCL6
highly expressed in no responsive ABC-DLBCL patients after Ibrutinib treatment. And in vitro experiment was carried out to validate if
BCL6
inhibitor can enhance the sensitivity of Ibrutinib in C481S BTK HBL-1 cells. FX1, a
BCL6
inhibitor, destroyed the formation of
BCL6
repression complex and suppressed ABC-DLBCL cell lines with IC
50
of 35 uM [
31
]. FX1 used in our study was lower than its IC
50
, which can increase sensitivity of Ibrutinb in C481S BTK HBL-1 cells. Thus, our finding that
BCL6
may be involved in drug resistance is consistent with previous studies. What is more,
BCL6
maybe the potential target to improve Ibrutinib sensitivity in C481S BTK HBL-1 cells.
BCL6
inhibits expression of various target genes via binding gene promoters.
BCL6
not only destroys interactions between T and B cells by
CD80
and PD-L1 but also inhibits B cell differentiation across decreasing expression of
PRDM1
and
IRF4
[
32
–
37
]. Above studies are consistent with the upregulation of
BCL6
, and downregulation of other hub genes in our present study.
PRDM1
/
BLIMP1
encodes a transcriptional repressor, which is necessary for differentiation of B cells into plasma cells [
38
]. Studies reported that
PRDM1
acts as a tumor suppressor gene in ABC-DLBCL in vivo mouse models [
39
,
40
]. As previously described,
PRDM1
is frequently inactivated by genetic alterations, including genetic deletions or mutations or transcriptional repression in ABC-DLBCL [
38
,
41
]. Parekh et al. found that different genetic alterations within
PRDM1
had adverse prognostic factors [
42
]. And inactivation of
PRDM1
can upregulate expression of
C-MYC
and downregulate expression of
p53
pathway molecule in ABC-DLBCL [
42
,
43
]. These results suggest that inactivation of
PRDM1
is closely linked to development of ABC-DLBCL.
IRF4
was essential for regulating gene transcription and mitochondrial homeostasis in plasma cells [
44
].
IRF4
activates or is repressed by
BCL6
, and co-expresses with
PRPM1
affecting plasma cells development [
45
–
47
]. However, most studies found that it does not express
PRDM1
protein though the presence of
IRF4
in ABC-DLBCL, suggesting other regulatory mechanisms between them [
40
]. Abnormal expression of
IRF4
is linked to several blood malignancies. For example, expression of
IRF4
is related to poor survival outcomes in peripheral T-cell lymphoma and chronic lymphocytic leukemia (CLL) [
48
,
49
]. What is more, studies showed
IRF4
dysregulation is associated with resistance to immunomodulatory compounds in Waldenström’s macroglobulinemia and myeloma [
50
,
51
]. A previous study has reported that Ibrutinib downregulates
IRF4
and consequently synergizes with lenalidomide in killing ABC DLBCL [
52
]. Another study indicated mutation of
IRF4
may explain the rel/ref phenotype of ABC-DLBCL [
5
]. Therefore, the role of
IRF4
in ABC-DLBCL need further explore.
Lin et al. identified that upregulation of miR-30 family can directly downregulate
BCL6
in B-lymphocytes and lymphoma cells [
53
]. Current studies found miR-30 family played a significant role in various tumors. Zhang et al. proved miR-30d could inhibit autophagy thereby promoting cell apoptosis [
54
]. The higher expression of miRNA-30c had better outcome with tamoxifen treatment in breast cancer [
55
]. Another investigation found that overexpression of miR-30b and miR-30c have better outcome after TKIs treatment in non-small cell lung cancer [
56
]. Interestingly, miR-30 family is considered as oncogenic miRNA, too. For instance, Gaziel-Sovran et al. reported that miR-30b and miR-30d promoted invasion of melanoma cells leading to generated
IL10
and reduced immune cells activation and recruitment [
57
]. Taken together, miR-30 family has complex functions in various cancers. However, the role of miR-30 family in Ibrutinib resistance of ABC-DLBCL has not been reported. Our study showed that miR-30 family may mediate Ibrutinib resistance in ABC-DLBCL, which is worthy of further exploration.
