CCGG deletion (rs201074739) in CD33 results in premature termination codon and complete loss of CD33 expression: Another key variant with potential impact on response to CD33-directed agents

Acute myeloid leukemia (AML) remains difficult to treat disease. While initial complete remission (CR) rates are >80%, overall survival remains unacceptably low due to high relapse rates even with intensive and myeloablative therapies. CD33 is a promising cell surface antigen target for immunotherapy in AML as it is expressed in AML blasts of 85–90% of all patients and upon binding to anti-CD33 antibodies the CD33-antibody complex is rapidly internalized^1–3. Gemtuzumab ozogamicin (GO), a CD33-antibody conjugated to DNA damaging cytotoxin, calicheamicin has been recently approved by FDA for treatment of CD33 positive AML^4,5. Encouraged by the results of GO, several new CD33 directed therapeutics such as Fc-engineered unconjugated antibodies (BI 836858 [mAb 33.1]), antibody drug conjugates (ADC) (SGN-CD33A [vadastuximab talirine], IMGN779, AVE9633), radio-immunoconjugates (²²⁵Ac-lintuzumab), bi- and trispecific antibodies (AMG330, AMG673, AMV564, 161533 TriKE fusion protein), and chimeric antigen receptor (CAR)-modified immune effector cell, are currently at different stages of development⁶.

Expression of CD33 on leukemic cells can impact the therapeutic efficacy of CD33-directed agents including GO. We have recently reported a splicing SNP (rs12459419 C>T) in CD33 that results in loss of exon 2 producing a short-form of CD33 (CD33-D2) lacking IgV domain^7,8. This exon produces the most immunogenic domain of CD33 and is recognized by gemtuzumab as well as the antibodies currently being used for immunophenotyping. Patients with CC genotype for rs12459419 that codes for full length CD33 benefit from addition of gemtuzumab where as patients with CT/TT or TT/TT show no benefit from gemtuzumab addition to AML chemotherapy⁷.

We present a case of a 68-year-old female AML patient following induction therapy that surprisingly had no detectable expression of CD33 on CD34+ progenitor cells, monocytes and neutrophils was observed in flow cytometric analysis of bone marrow aspirates (Figure 1A). We utilized p67.6 CD33 antibody that targets IgV domain of CD33. The patient proceeded to hematopoietic cell transplantation (HCT) without detectable residual disease. The specimens obtained post-HCT were re-evaluated by flow cytometry and demonstrated normal levels of CD33 on monocytes, progenitor cells and neutrophils (Figure 1B) reflecting cells from the donor. Two months post-transplant the patient relapsed and the leukemia cells again had no detectable expression of CD33 when measured by flow (Figure 1C).

Figure 1.

Flow cytometry dot plot analysis of bone marrow samples obtained at different time points from 69-year-old female patient with acute myeloid leukemia. A) Non detectable levels of CD33 in cells from bone marrow aspirate obtained prior to hematopoietic stem cells transplantation (Pre-HSCT). B) Post-allogeneic stem cell transplant in remission sample (Post HSCT). The cells from the donor show expression of CD33 in both the monocyte and the CD34⁺ progenitor cells. C) Sample obtained at relapse shows non-detectable levels of CD33 in AML cells. CD34⁺ cells are shown in red and the monocytes (identified by CD45 and side scatter) in dark green. D) Sequence of control and AML patient sample showing homozygous CCGG 4 bp deletion (-/CCGG: genotype) for rs201074739. Comparison of protein sequence of full length and truncated CD33 protein caused by presence of CCGG deletion.

The patient DNA obtained prior to transplant was genotyped for rs12459419 (C>T) splicing SNP affecting exon 2 and showed presence of CC genotype, that codes for the full length CD33 protein. To identify the potential cause for the loss of CD33 expression observed in pre-transplant and post relapse specimens we also genotyped genomic DNA for additional SNPs (rs1803254 a 3’UTR SNP, rs35112940 and rs61736475 - nonsynonymous coding SNPs and rs201074739, a CCGG deletion) using Taqman allele discrimination assays from Applied Biosystems. Additionally, all the coding CD33 exons along with the flanking intronic region, 3’ and 5’ UTRs were sequenced using primers and conditions reported previously to identify any other potential variant ^9,10. The patient was homozygous for only the rs201074739 CCGG deletion (−/− genotype) which was confirmed by sequencing as shown in Figure 1D. Genomic DNA from bone marrow aspirate, CD3⁺, and CD13⁺ sorted cells from the patient during remission were consistent with all samples showing homozygous deletion of CCGG for rs201074739 (−/−). The rs201074739, 4 bp CCGG deletion occurs in exon 3 of CD33 and alters the reading frame creating premature termination codon (PTC). The truncated protein produced due to PTC codes for only 159 amino acids in contrast to full length CD33 with 364 amino acids (Figure 1D). CD33 mRNA levels were quantitated in pre-transplant cells using methods previously described⁷, the patient had very low levels of CD33 as compared to patients without CCGG deletion (CCGG/CCGG) and without splicing SNP (CC for rs12459419), supplementary Figure 1A. Within GTEx database (https://gtexportal.org/home/) also rs201074739 was significantly associated with the CD33 mRNA levels (Supplementary Figure 1B).

Nonsense mediated decay (NMD) pathway acts as a surveillance mechanism within a cell that targets mRNA transcripts with premature termination codons (PTC) for degradation ^11,12. We tested whether the mRNA transcript with CCGG deletion are targeted for degradation by NMD. Given NMD pathway relies on translation it has been shown that blocking protein synthesis can inactivate NMD thereby stabilizing mRNA transcripts with premature termination codon¹³. Viable cells from the patient were not available to assess whether CD33 transcript with CCGG del is degraded via NMD pathway. We screened several AML cell lines for presence of CD33 SNP and none were positive for CCGG deletion. We screened 1000 genome database and identified sample NA07048 from CEU HapMap panel (European ancestry) to be heterozygous -/CCGG for rs201074739. Overall rs201074739 occurs with a very low allele frequency of 0.01. We obtained lymphoblast cell lines from the HAPMAP family trio 1341 from Coriell cell repositories (https://www.coriell.org) and genotyped for rs201074739. The father-NA07034 was heterozygous for rs201074739 with genotype -/CCGG, mother-NA07055 was homozygous for reference allele with no deletion (CCGG/CCGG genotype) and child -NA07048 was confirmed to be heterozygous for deletion with -/CCGG genotype (Figure 2A). Even though lymphoblast cell lines have low CD33 expression, we were able to see the impact of the presence of CD33 SNP on CD33 cell surface expression levels (Cell lines 7034 and 7048 had very low CD33 as compared to 7055 expression Supplementary Figure 2). Thus though LCLs might not be ideal but were the best model available to study the NMD associated with this 4 bp deletion variant in CD33. Given the NMD pathway for degradation of mRNAs with PTCs should be consistent across cell types, we used these three cell lines to test for CD33 transcript carrying PTC for degradation via NMD. Protein synthesis inhibitor cycloheximide (CHX) blocks translation and thus inhibits NMD resulting in stabilization of the PTC containing transcripts and was used to evaluate these cell lines. Briefly all the three-lymphoblast cell lines NA07034, NA07055, and NA07048 cell lines were maintained in an RPMI 1640 medium supplemented with 20% FBS as per manufacturer’s instructions (Coriell cell repositories). Cell lines were treated with 30 μg/ml CHX or ethanol for control (Santa Cruz Biotechnology, Inc.) for 4 hr followed by CHX free medium for additional 4 hr shown in figure 2. Total RNA was isolated using Trizol and cDNAs were first synthesized from total RNA using TaqMan Reverse Transcription Reagents (Life Technologies). CD33 transcript levels were quantified and compared to 18S rRNA as endogenous internal control. All assays were performed three times in triplicate.

Figure 2.

A) Family trio with a father and son being carriers of CCGG 4 bp deletion (-/CCGG: genotype) for rs201074739. Inhibition of translation with CHX in a -/CCGG lymphoblast cell lines NA07048 (B) and NA07034 ( C) shows increase in CD33 mRNA levels indicating CHX mediated inhibition of non-sense mediated decay is resulting in increase in CD33 mRNA transcripts with PTC. D) Inhibition of translation with cycloheximide (CHX) in a CCGG/CCGG (NA07055) lymphoblast cell line has no significant impact on CD33 transcript levels. Removal of CHX reverses mRNA levels similar to base line indicating reinitiation of translation triggers NMD of the transcript containing PTC only in NA07048 and NA07034 (B and C) but not in NA07055 (D).

4 hr-CHX treatment of NA07048 (-/CCGG) cell line resulted in significant increase (2.8-fold) in CD33 mRNA levels (Figure 2B). Similar results were obtained for the paternal cell line (NA07034), also heterozygous for the deletion (Figure 2C). However, no significant difference in the CD33 transcript with 4 hr CHX treatment was observed for maternal cell line-NA07055 (CCGG/CCGG genotype), Figure 2D. To determine whether the PTC CD33 mRNA was specifically regulated by protein synthesis, all three-cell lines were either untreated or treated with CHX for 4 h followed by removal of CHX and further incubation in drug-free medium for an additional 4 h. CD33 expression levels in CHX-treated NA07048 and NA07034 returned to pre-treatment levels 4 h after removal of CHX indicating reinitiation of translation triggers NMD and abrogates the stabilization of these mRNAs (Figure 2B and C). In contrast the maternal cell line NA07055 showed no difference in levels at 4hr CHX treatment of 4 hr post removal of CHX (Figure 2D). These results suggest CD33 mRNA containing PTCs were stabilized by CHX and upon removal of CHX, NMD pathway mediated degradation of CD33 is resumed.

In summary, our results show an AML patient with undetectable CD33 expression in pre-BMT bone marrow aspirate and at relapse. The patient was homozygous CC for the splicing SNP present in exon 2, but homozygous for 4 bp CCGG deletion (−/−) for rs201074739 within exon 3. CCGG deletion changes reading frame and creates a premature termination codon thus resulting in the undetectable levels of CD33 observed in this patient. So far, to the best of our knowledge, this is the first report of an AML patient homozygous for the CD33 rs201074739 SNP. Using in vitro systen, we showed for the first time that CD33 transcripts carrying the rs201074739 variant allele were subject to NMD-dependent degradation. Our results provide further rationale for the development of a complete spectrum of all potentially detrimental CD33 SNPs as biomarkers for CD33-directed therapies for treating AML patients.