IKZF3 amplification occurs frequently in breast cancer with a prevalence of 12-15%
To systemically investigate commonly amplified genes in human breast cancer, we queried 4 major databases deposited in cBioPortal [18], including the Breast Invasive Carcinoma TCGA PanCancer data [21], the AACR Project GENIE Consortium[22], the Metastatic Breast Cancer DFCI-Profile cohort [23, 24], and the TCGA METABRIC cohort[25, 26]. We confined our query to specifically include patients with invasive ductal breast carcinoma (IDC). From the aforementioned databases in cBioPortal, the frequency of amplified genes was listed and ranked in each database. We used an arbitrary cutoff of 10% frequency for gene amplification, i.e. only genes with a ≥ 10% amplification frequency were selected for further analysis. We discovered that there was a vast difference between databases of numbers of gene amplification reported with ≥10% frequency. In the TCGA METABRIC and PanCancer cohorts, the numbers of amplified genes with ≥10% frequency were 1579 and 476, respectively, while in AACR genie and the DFCI cohort (which was curated under the AACR-genie program), the numbers of amplified genes were 19 and 3, respectively. This may be related to the threshold of gene amplification reporting, as AACE-genie guidelines state only CNAs with a ploidy of 5 or higher are reported as gene amplification[27]. Nevertheless, the 3 genes from the DFCI cohort (IKZF3, CCND1, ERBB2 ) were consistently reported as amplified in the all 4 cohorts (Fig. 1A, table 1). An additional 11 genes (COL22A1, FGFR1, SETDB1, FGF19, UBR5, FGF4, NSD3, MYC, TG, PDE4DIP, FGF3) were reported as amplified in the AACR-genie, TCGA-METABRIC and TCGA-Pancancer cohorts but not in the DFCI cohort. Therefore we concluded that IKZF3, CCND1, ERBB2 was consistently reported as amplified in human breast cancers, regardless of different cutoff criterion.
Further analysis into table 1 revealed several interesting observations: the 11q13.3 arm harbored CCND1, FGF3, FGF4, FGF19 that were consistently amplified in the 3 cohorts (TCGA METABRIC, TCGA Pancancer, AACR-genie). Other hotspots included chromosome 8, (FGFR1, NSD3 on 8p11.23, and MYC, COL22A1, TG, UBR5 in 8q24), chromosome 1 (PDE4DIP, SETDB1 on 1q21.3), and chromosome 17 (ERBB2, IKZF3 on 17q12). The 3 genes IKZF3, CCND1, ERBB2 were consistently amplified across all four cohorts analyzed. CCND1 encodes for cyclin D1, which is well characterized for its role in cell cycle. The role of cyclin D1 amplification in breast cancer has been well investigated and associated with a poor outcome in the METABRIC cohort [28]. Since cyclin D1 is an important element of the cell cycle, CDK4/6 inhibitors are theoretically effective in these group of patients. CDK4/6 inhibitors in luminal breast cancers have revolutionized the standard of care; however, amplification of cyclin D1 was not associated with a differential response to palbociclib in the PALOMA-1 trial[29]. ERBB2 encodes for HER2, and anti-HER2 therapeutics have drastically altered the nature course of HER2 amplification breast cancers [30, 31]. Compared to ERBB2 and CCND1, the role of IKZF3 has never been studied in a large-scale breast cancer patient cohort. Therefore, we elected to further investigate the role of IKZF3 amplification in clinical outcomes of breast cancer. To visualize the relative location of IKZF3 to ERBB2, we illustrated the proposed ERBB2 amplicon[15] on chromosome 17 using the Genome Data Viewer (https://www.ncbi.nlm.nih.gov/genome/gdv/) (Fig. 1B). Since IKZF3 belonged to the IKAROS transcriptional family with five members (IKZF1/2/3/4/5), we investigated the global expression levels in human cancers. Using the Oncomine database[16], we compared the expression levels between various human cancers. In general, only IKZF1 was more differentially expressed in breast cancer, while IKZF3 was differentially expressed in lymphoma and leukemia (Fig. 1C). Focusing on the BRCA cohort in TCGA, we observed that IKZF3 was generally underexpressed in comparison to the other members of the IKAROS family (Fig. 1D, E). Our findings suggest that IKZF3 in general is not highly expressed in breast cancer, which suggests amplification of IKZF3 might have a more substantial effect due greater fold changes in expression.
Table 1
|
|
metabric
|
PanCancer
|
genie
|
DFCI
|
CCND1
|
11q13.3
|
16.30%
|
14.20%
|
16.20%
|
13.70%
|
COL22A1
|
8q24.23-q24.3
|
24.20%
|
12.90%
|
21.90%
|
|
ERBB2
|
17q12
|
17.80%
|
13.50%
|
13.80%
|
13.60%
|
FGF19
|
11q13.3
|
16.30%
|
13.80%
|
16.10%
|
|
FGF3
|
11q13.3
|
15.60%
|
13.80%
|
15.30%
|
|
FGF4
|
11q13.3
|
15.70%
|
13.80%
|
15.70%
|
|
FGFR1
|
8p11.23
|
13.80%
|
11.50%
|
11.50%
|
8.80%
|
IKZF3
|
17q12-q21.1
|
15.50%
|
13.30%
|
13.50%
|
12.40%
|
MYC
|
8q24.21
|
28.60%
|
18.50%
|
10.40%
|
5.50%
|
NSD3
|
8p11.23
|
14.20%
|
11.80%
|
11.50%
|
|
PDE4DIP
|
1q21.2
|
15.50%
|
10.20%
|
13.90%
|
|
SETDB1
|
1q21.3
|
18.30%
|
10.20%
|
16.50%
|
|
TG
|
8q24.22
|
25.40%
|
13.70%
|
22.50%
|
|
UBR5
|
8q22.3
|
23.70%
|
12.90%
|
21.40%
|
|
IKZF3 amplification is highly associated with HER2 amplification and with worse outcome in breast cancer
To further delineate the role of IKZF3 in the clinical outcome of breast cancer patients, we performed in-depth analysis of the METABRIC database. Of the 1865 patients with IDC, 1660 patients had CNV analysis, in which 258 patients (15.5%) were classified as IKZF3 amplification (IKZF3-amp) and 1402 (84.5%) as non-amplified (IKZF3 non-amp). Overall survival was significantly shorter for IKZF-amp (median 96.9 months, 95% CI: 79.97 - 124.13) versus IKZF3 non-amp (median 163.7 months , 95% CI: 152.07 - 174.80, logrank test p-value 0.000000729) (Fig. 2A). IKZF3-amp patients had several clinical features significantly different compared to IKZF3-non amp patients (summarized in Supplemental 1: Table S1). These included: higher rates of HER2 positive status, lower rates of ER/PR positivity, younger age, higher rates of receiving chemotherapy, more advanced stages (higher proportion of stages III-IV), higher rates of mastectomy. Interestingly, 79.22% of IKZF3-amp patients were HER2 status positive (183/258), while an overwhelming 97.24% were HER2 negative in the IKZF3-non amp group (Fig. 2B). ERBB2 was the top differentially expressed gene when compared between IKZF3-amp and non-amp (Fig. 2B). A total of 31 genes were highly differentially expressed in the IKZF3-amp group of more than 2-fold (log ratio≥1) (Supplemental Table S2). In this group, ERBB2, GRB7, PGAP3, PPP1R1B, MIEN1, PNMT, FBXL20, CDK12 were all on 17q12, suggesting a linkage disequilibrium relationship with IKZF3. For instance, expression of GRB7, the gene adjacent to ERBB2, has been reported to be highly correlated with HER2 amplification status [32].
To further confirm this observation, we pooled the TCGA-PanCancer cohort and the TCGA-metabric cohort, resulting in a total of 360 patients with IKZF3 amplification and 2283 patients without. Overall survival was still significantly shorter for IKZF-amp (median 101.3 months, 95% CI 87.10 - 132.20) versus IKZF3 non-amp (median 163.2 months , 95% CI 152.07 - 174.50, logrank test p-value 0.00000061) (Fig. 2d). Since most of the samples were early breast cancer (>95% stage I or II), surgery was the mainstay treatment. The relapse free survival still showed a significant benefit in IKZF3-non amp patients (Fig. 2E). To test whether IKZF-amp would have an adverse impact on survival independently of HER2 amplification, we compared patients with and without HER2 amplification in the IKZF3-amp subgroup. There was a trend favoring patients without Her2 amplification with better overall survival, although the difference was not statistically significant (HER2 amp versus HER2 non-amp, overall survival 87.1 months versus 129.2 months, log rank p=0.0751) (Fig. 2F)
IKZF3 expression is decreased after anti-HER2 treatment
Our studies suggest a possible role of IKZF3 in HER2 (+) breast cancer treatment. We then investigated whether the expression of IKZF3 would be modulated by anti-HER2 treatment. With an aim to investigate the changes of IKZF3 in real world patient based studies, we queried the GEO dataset GSE114082. This study quantified RNA-seq expression levels before and after a single dose of trastuzumab in 17 HER2-positive breast cancer patients[33]. Interestingly, both IKZF3 and ERBB2 major transcript levels decreased after treatment of anti-HER2 trastuzumab (Fig. 3A). We then performed pathway analysis using the ingenuity pathway analysis (IPA) software. RNA-seq analysis of the dataset GSE114082 revealed top enriched pathways : intrinsic prothrombin activating pathway, cytokine signaling, atherosclerosis signaling, serotonin signaling, and LXR/RXR signaling (Fig. 3B). We then analyzed differentially expressed genes with DAVID (with filter options selected: “OMIM_DISEASE “, “GOTERM_BP_DIRECT”, “GOTERM_CC_DIRECT”, “GOTERM_MF_DIRECT”, “BIOCARTA”, “ KEGG_PATHWAY “), in which top ranked pathways included: Olfactory transduction, Intrinsic Prothrombin Activation Pathway, ABC transporters, G-protein coupled serotonin receptor activity, Chemical carcinogenesis (Table 2 ). Interestingly, intrinsic pathway of coagulation was enriched in both IPA and DAVID pathway analysis approaches. Differentially expressed genes in this pathway included F8, PROC, F9, F10, F12, F11, COL4A3, KNG1, which we confirmed were elevated from around 3 fold to 7.6 fold after treatment by trastuzumab, with the exception of F12 and PROC gene which were downregulated (Fig. 3C). To summarize, we discovered that IKZF3 expression decreased synchronously with ERBB2 after trastuzumab treatment. This suggested that HER2- amplification clones that also co-expressed (or co-amplified ) IKZF3 might be eradicated after trastuzumab treatment. Our data also suggested that treatment of trastuzumab, and decrease of HER2/IKZF3, was associated with modification of several transcriptional pathways including the coagulation pathway.
Table 2
Enrichment score
|
Term
|
Count
|
P-Value
|
FDR
|
Category
|
85.31
|
Olfactory transduction
|
187
|
0.000
|
0.000
|
KEGG_PATHWAY
|
2.72
|
Intrinsic Prothrombin Activation Pathway
|
8
|
0.000
|
0.028
|
BIOCARTA
|
2.26
|
ABC transporters
|
9
|
0.022
|
0.374
|
KEGG_PATHWAY
|
2.01
|
G-protein coupled serotonin receptor activity
|
7
|
0.006
|
0.548
|
GOTERM_MF_DIRECT
|
1.72
|
Chemical carcinogenesis
|
17
|
0.001
|
0.060
|
KEGG_PATHWAY
|
DepMap analysis on effect of IKZF3 gene disruption suggest dependence on IKZF3 in HER2 and triple-negative breast cancer cell lines
To further analyze whether breast cancer cells, especially HER2 amplified cells, exert dependence on IKZF3, we queried the DepMap Portal (https://depmap.org/portal/depmap/) [34]. By interrogating the CRISPR DepMap 21Q3 Chronos dataset, focusing on ductal breast cancer cell lines, we identified cell lines that demonstrated dependency on IKZF3 (Fig. 4A). In this analysis, each cell line was tested for their dependence to IKZF3 by a genome wide CRISPR knockout screen. Interestingly, cell lines showing mild IKZF3 dependency (Gene effect score < -0.2) were exclusively TNBC (triple negative breast cancer) (HCC1143、MDAMB157、SUM1315MO2) and HER2 amplification (UACC893、HCC202、HCC1419 ) cell lines. As a comparison, we also examined ERBB2, which showed dramatically more cell lines dependency and markedly stronger dependency (Fig. 4B) However, cell lines dependent on ERBB2 included not only TNBC and HER2 BC cell lines, but also HR(+) cell lines (T47D, ZR751, and others) demonstrating that ERBB2 is a strong oncogenic driver that elicits significant oncogene addiction. This analysis suggested that HER2 and TNBC breast cancer cell lines are more likely to be dependent on IKZF3 signaling.