Trisomy 12 CLLs progress through NOTCH1 mutations

B-cell chronic lymphocytic leukemia (CLL) is the most common adult leukemia in Western societies.¹ CLL cases can be subgrouped into two types, aggressive or indolent, defined as cases that express high levels of Zeta-chain-associated protein kinase 70 (ZAP70) and unmutated immunoglobulin heavy-chain variable-region genes (IGHV), or low to negligible ZAP70 and mutated IGHV. Chromosomal aberrations can be identified in more than 80% of patients.¹ The most frequent genetic alterations include deletion/ inactivation of 13q14 (450%), deletion of 11q22–23 (18%), trisomy of 12 (12–16%) and deletion 17p (7–10%).¹

Two recent studies reported whole-genome sequencing of CLL samples and found NOTCH1 is the most frequently mutated gene in CLL,^2,3 although we found a mutation frequency of only 3.5% in a large cohort of CLL.⁴

Our recent report showed that NOTCH1 mutations in CLL associate with trisomy 12.⁴ We found 41.9% NOTCH1 mutation frequency in aggressive trisomy 12 CLL cases, suggesting that activation of NOTCH1 has a critical role in IGVH unmutated/ ZAP70 + trisomy 12 CLL.⁴

NOTCH1 encodes a class I transmembrane protein functioning as a ligand-activated transcription factor.^5,6 Upon ligand binding, Notch1 undergoes several proteolytic cleavages resulting in translocation of the Notch1 intracellular domain (ICN) to the nucleus where it has an important role in cell differentiation, proliferation and apoptosis, leading to transcriptional activation of multiple target genes, including MYC.⁷ ICN contains PEST domain targeting ICN for ubiquitinylation and degradation.^5,6 Almost all NOTCH1 mutations in CLL are represented by the two base deletion frame-shift, resulting in a truncated constantly active protein lacking the C-teminal PEST degradation domain.^2,3

On the basis of our previous findings, we investigated whether NOTCH1 mutations are early events in trisomy 12 CLL, or these mutations are associated with CLL progression. Mutations were most commonly observed in 100% of cells with one WT allele and one mutant allele, but we also found samples with different mutation frequencies.⁴ Looking for change in mutation frequencies, we sequenced serial samples of 78 trisomy 12 CLL patients, and found 10 informative cases (Table 1). First, we studied two samples from each patient taken as far as possible. Patients were than subgrouped in two categories: patients with constant NOTCH1 mutation rate and patients with changes in NOTCH1 mutation rate. Only samples showing changes in NOTCH1 mutation rate were considered informative. Clinical data were obtained to verify the disease progression and middle time points of these patients were tested to verify changes in NOTCH1 mutation rate during progression. In these cases mutation frequency varied from 100% WT alleles (both alleles were WT) to 10% WT allele and 90% mutated allele (in most cells both alleles were mutated) (Supplementary Figure 1).

Table 1.

Detailed description of informative CLL samples showing that NOTCH1 mutations associate with CLL progression

Patient no.	Sample date	NOTCH1 Mut	Sample-ID	WT%	Mut%	WBC (109/l)	Spleen (cm)	B2M (mg/dl)	%ZAP	%VH
11	13/7/2009	ΔCT	51V	90	10	30.9	0	1.5	10.4	99.7
	16/5/2011	ΔCT	1069	60	40	126.3	0	1.3
15	9/4/2002	ΔCGC	8Y	90	10	43.7	3	2.1	25.1	100
	Treated 21/1/2003
	31/7/2003	ΔCGC	1046	90	10	ND	2	3
	14/12/2005	ΔCGC	1047	65	35	439	ND	8.2
	Treated 11/4/2006–31/8/2006
	1/11/2006	ΔCGC	1048	100	0	3.5	ND	1.9
56	7/4/2009	ΔCT	1024	100	0	24.8	2	2.8	91.9	100
	Treated 1/1/2009–1/9/2009
	9/11/2010	ΔCT	1137	95	5	5.1	ND	2.7
	7/6/2011	ΔCT	1023	80	20	35.5	2	3.5
	8/7/2011	ΔCT	1111	80	20	53	2	3.8
65	8/8/2006	ΔCT	59R	50	50	21.9	Edge palpable	2.5	92.4	99.6
	30/10/2007	ΔCT	6V	25	75	87.2	ND	3.5
	Treated 17/6/2008–11/9/2008
	21/11/2008	ΔCT	1013	50	50	5	ND	3.6
	15/6/2009	ΔCT	1014	25	75	45.4	5	5.1
	Treated 1/8/2009–1/10/2009
	30/11/2009	ΔCT	1118	50	50	5.4	ND	3.9
	Treated 14/7/2010–1/11/2010
	11/1/2011	ΔCT	1119	100	0	ND	ND	ND
69	31/7/2007	ΔCT	50V	50	50	26.5	ND	2.9	97.7	100
	25/1/2008	ΔCT	1121	25	75	71.5	5	6.4
36	17/1/2006	ΔCT	33V	50	50	56.8	5	5.1	83	100
	Treated 18/1/2006–7/1/2006
	9/10/2008	ΔCT	38M	100	0	3.4	0	2.5
52	22/7/2005	ΔCT	5E	100	0	4.8	0	2.5	67.6	Unmuted
	Treated 2007
	5/3/2008	ΔCT	5M	50	50	13.2	Enlarged	7.3
77	27/1/2006	ΔCT	49EM	75	25	15	0	1.9	Pos	Unmuted
	21/1/2009	ΔCT	49M	25	75	216	0	3.7
66	21/4/2009	ΔCT	1139	100	0	4.6	ND	ND	73.1	100
	28/7/2010	ΔCT	1015	10	90	18.6	ND	ND
28	28/2/2007	ΔCT	57R	100	0	88.3	Not palpable	ND	61.1	99.6
	Treated 9/1/2007
	22/10/2008	ΔCT	1006	80	20	8.5	Not palpable	ND
	15/12/2010	ΔCT	1132	65	35	48.2	Not palpable	ND

Abbreviations: B2M, beta(2)-microglobulin; CLL, chronic lymphocytic leukemia; MUT%, ND, not determined; NOTCH1 mutated percentage calculated on both alleles; VH%, percentage of mutation in the variable-heavy region of immunoglobulin chain genes; WBC, white blood count; WT%: NOTCH1 wild-type percentage calculated on both alleles; ZAP70: Zeta-chain-associated protein kinase 70.

Four cases (28, 52, 56 and 66) did not show mutations at the first time points, however, increased mutation frequency was evident at later time points. In all these cases increases in NOTCH1 mutation frequencies were associated with progression of the disease.

Patient 56 showed increase in the mutant allele from 5 to 20%, while white blood cell count (WBC) increased from 5.1 to 53×10⁹/l, and beta(2)-microglobulin (B2M) increased from 2.7 to 3.8 mg/dl. Clinical data supported CLL progression between the time points provided. Patient was first diagnosed in 2001. In April 2009, patient was treated with flurabine and rituximab and showed 0% of NOTCH1 mutation rate. After successful treatment ended in September 2009, NOTCH1 mutation rate rise to 5% as observed in 2010. Patient relapsed and progressed with increase of NOTCH1 mutation to 20% in 2011. In August 2011, patient reinitiated fludarabine and rituximab treatment.

Patient 66 showed increase in the mutant allele from 0 to 90% while WBC increased from 4.6 to 18.6 × 10⁹/l. Patient was treated before the first time point achieving complete response and showed wild-type (WT) NOTCH1 in April 2009. However, contemporaneously with raising of percentage of NOTCH1 mutation rate to 90%, patient developed progressive disease. Bone marrow biopsy revealed presence of lymphocytes constituting 50–60% of overall cellularity, 39% of them having immunophenotype consistent with CLL at flow cytometry evaluation.

Patient 52 showed increase in the mutant allele from 0 to 50%, while WBC increased from 4.8 to 13.2 × 10⁹/l, and B2M increased from 2.5 to 7.3 mg/dl. Patient was initially treated in 2004 and achieved partial response. In 2005, NOTCH1 gene sequence was WT. In 2007, indolent relapse of disease with hemolytic anemia occurred and patient was treated with rituxan. In April 2008, patient relapsed again, disease progressed and NOTCH1 mutation rise to 50%. Consecutively, patient was treated with oxiplatin, fludarabine, cytarabine and rituximab.

Five other cases (11, 15, 65, 69 and 77) also showed increased mutation frequency with disease progression, although in these cases mutations were already present in the first time point samples. Interestingly, in three patients (15, 65 and 36) decreased mutation frequency was associated with treatment response. Patient 15 showed decrease in the mutant allele from 35 to 0%, while WBC decreased after treatment with fludarabine, rituximab and cyclophosphamide from 439 to 3.5 × 10⁹/l, and B2M decreased from 8.2 to 1.9mg/dl. Patient 36 showed decrease in the mutant allele from 50 to 0%, while WBC decreased after treatment with cyclophosphamide, fludarabine, alemtuzumab and rituximab from 56.8 to 3.4 × 10⁹/l, and B2M decreased from 5.1 to 2.5mg/dl.

To investigate the molecular consequences of the expression of constantly active Notch1 protein in trisomy 12 CLLs, we performed genome-wide Affymetrix array analysis by comparing NOTCH1 WT and NOTCH1-mutated samples. To insure reliability of the results all chosen samples were IGVH unmutated/ZAP70 + , and >50% of cells showed trisomy 12 (Supplementary Table 1). Ten of ten NOTCH1 WT samples and six of seven NOTCH1-mutated samples showed tight clustering (Supplementary Figure 2A), the remaining NOTCH1-mutated sample (Mut1) showed only partial clustering (Supplementary Figure 2A). Among 20 most upregulated genes in NOTCH1-mutated samples, we did not find any genes with known oncogenic function. However, 10 of the 20 most downregulated genes in NOTCH1-mutated samples have known tumor suppressor or pro-apoptotic function (Table 2). Among these downregulated genes, we found three members of the FOS gene family associated with apoptotic cell death in CLL⁸(c-FOS, FOSB and FOSL2), CDKN1A (p21), a p53-dependent cell cycle inhibitor,⁹ and NR4A3 (NOR1), a known inducer of apoptosis in lymphoid cells¹⁰ (Table 2). Microarray data were confirmed by real-time reverse-transcription PCR for the following genes: CDKN1A (p21), c-FOS and FOSB (Supplementary Figure 2B). Thus, we concluded that activated Notch1 inhibits multiple tumor suppressors in trisomy 12 CLLs.

Table 2.

Ten out of 20 most downregulated genes in NOTCH1-mutated samples have known tumor suppressor or pro-apoptotic function.

Gene name	Reference	P-value	FC	Function
CDKN1A	Harper et al.9	0.00457	2.43	Tumor suppressor
FOS	Pekarsky et al.8	0.00589	3.62	Pro-apoptotic
FOSB	Pekarsky et al.8	0.01604	6.56	Pro-apoptotic
DUSP10	Tanoue et al.11	0.02811	2.30	Anti-proliferative
IFIT2	Stawowczyk et al.12	0.03051	2.19	Tumor suppressor
NR4A3	Mullican et al.10	0.03151	2.52	Pro-apoptotic
EGR1	Huang et al.13	0.03359	3.60	Pro-apoptotic
KLF4	Shields et al.14	0.03436	3.01	Anti-proliferative
FOSL2	Pekarsky et al.8	0.03591	2.10	Pro-apoptotic
IFIT3	Xiao et al.15	0.05028	3.08	Anti-proliferative

Abbreviation: FC, fold change.

As recent studies identified NOTCH1 as the most mutated protein-coding gene in CLL,^2,4 it is important to determine whether NOTCH1 mutations are essential in the initiation of CLL, or whether these mutations are associated with progression of trisomy 12 CLLs. Here we found nine CLL cases showing increase of NOTCH1 mutation frequencies associated with severity of the disease including four cases showing WT NOTCH1 at initial time points. Activation of NOTCH1 appears to be involved in downregulation of tumor suppressor and apoptotic key factors, accelerating the progression of the disease. Taken together, these data indicate that NOTCH1 mutations do not cause CLL but rather associate with CLL progression leading to more aggressive form of the disease with poor outcome.

Supplementary Material

Supplementary Materials

Supplementary Figure 1. Examples of different NOTCH1 mutations rates in CLL samples.

Supplementary Figure 2. Microarray analysis of NOTCH1 WT and Mutated samples. A. Clustering of the samples. B. Real time RT-PCR validation of microarray data.