APOBEC3B Overexpression Linked to Tamoxifen Resistance

Reuben Harris, PhD

The enzyme APOBEC3B, which is overexpressed in about 50% of breast tumors, has been associated with resistance to treatment with tamoxifen in retrospective studies and xenograft models of ER-positive breast cancer.

Across these studies, overexpression of APOBEC3B was related to a worse prognosis, treatment resistance, and rapid tumor growth compared with low expression of the enzyme. These findings suggest that APOBEC3B could be a viable therapeutic target for preventing resistance to therapy and tumor growth, according to a presentation at the 2015 San Antonio Breast Cancer Symposium.¹

“High APOBEC3B expression levels correlate with poor outcomes for ER-positive breast cancer, including recurrence during tamoxifen treatment,” said lead investigator Reuben Harris, PhD, from the Masonic Cancer Center at the University of Minnesota. “Unlike many of these things that are driving tumors at a molecular level, this is a gain-of-function enzyme, it is not a loss-of-function that takes away DNA repair, such as BRCA.”

APOBEC is a family of proteins that have been associated with cancer genome mutations. Within this family, the APOBEC3B enzyme has specifically been implicated as a cause of mutations in breast cancer that lead to metastases, heterogeneity, and resistance to therapy. As expression of this enzyme increases, tumor mutational loads expand along with tumor evolution.

To further explore the role the enzyme, a retrospective study was conducted to assess APOBEC3B messenger RNA across tumor samples from patients with breast cancer.² In order to remove confounding factors, a cohort of patients was selected who did not receive adjuvant or neoadjuvant therapy (n = 829). Within this group, 633 patients had ER-positive, node-negative breast cancer and 196 had ER-negative disease.

In the ER-positive population, after 10 years of follow-up the risk of disease recurrence was 55% higher in patients with high expression of APOBEC3B (HR,&thinsp;1.55; 95% CI, 1.23-1.96; P&thinsp;<&thinsp;.001). Additionally, when APOBEC3B was highly expressed there was a 66% increase in metastases risk (HR, 1.66; 95% CI,&thinsp;1.26-2.17; P&thinsp;<.001) and a 68% increase in the risk of death (HR,&thinsp;1.68; 95% CI;&thinsp;1.25-2.24; P &thinsp;<.001).

“Over time, for both disease-free survival and overall survival, there's a significant difference between those who have high- and low-expression of this particular DNA mutating enzyme,” said Harris.

To further tease out the added risk, another retrospective study looked at the impact of APOBEC3B expression on the efficacy of tamoxifen.³ For this analysis, samples were selected from 285 hormone therapy-naive patients with ER-positive breast cancer who were receiving frontline tamoxifen. These samples were reviewed in two independent cohorts.

In both groups, high-levels of APOBEC3B were associated with an unfavorable prognosis. For those with high expression, the median progression-free survival was 7.5 versus 13.3 months for those with low expression (HR, 1.67; P = .0001).

“Those with the highest levels of APOBEC3B faired much more poorly than those with the low levels, with intermediate levels tracking between those,” said Harris. “This is a highly significant result. Those primary tumors with low APOBEC3B had a much longer progression-free survival.”

To confirm the findings in a more prospective manner, xenograft models for APOBEC3B expression were developed and treated using tamoxifen or a control. In general, in vitro cell growth was similar between models with low and high APOBEC3B; however, in vivo APOBEC3B overexpression was found to accelerate the development of tamoxifen-resistance.

In these early models, suppression of APOBEC3B was associated with a prolongation in response to tamoxifen (P <.05). Moreover, this response was durable and lasted for roughly 12 months, said Harris. These findings suggest that a treatment could be developed to block APOBEC3B and delay or prevent the development of treatment resistance.

In the control group that did not receive treatment, tumors with APOBEC3B high expression grew very rapidly, with death occurring at approximately 150 days. For those with low APOBEC3B expression, survival was prolonged to approximately 190 days.

At the 12-month data cutoff, mice treated with tamoxifen remained alive but had significantly larger tumors (~900 mm³), when APOBEC3B was overexpressed. These tumors were considered to have normal levels of APOBEC3B, said Harris. In a group with suppressed APOBEC3B, tumor growth was significantly delayed, resulting in smaller masses (~300 mm³).

“Without tamoxifen therapy, the tumors grow very rapidly into large tumors. In the presence of tamoxifen therapy, applied at day 100 for these animals, one can see that those with normal APOBEC3B in their cell-line developed resistance to tamoxifen therapy over time,” Harris said. “If we knock down this one single DNA mutating enzyme, one can see that the therapeutic durability is much more effective. Most of these animals did not develop resistance.”

In a second experiment, models were forced to overexpress APOBEC3B. In this group, similar findings were seen. Without treatment there was a quick progression. When therapy was applied, resistance developed quickly.

This was particularly true when the wild-type APOBEC3B enzyme was overexpressed, which has DNA deaminase activity, Harris said. In this group, there was instant tamoxifen resistance.

“If we overexpress the catalytic mutant, we don't change results at all. In the presence of therapy, resistance develops relatively rapidly. There are endogenous levels of APOBEC3B still in the system,” said Harris. “If we overexpress the wild-type enzyme, which does have DNA deaminase activity, one can see the effect of tamoxifen is almost zero.”

APOBEC3B-directed therapies are still in early preclinical development, to potentially capitalize on this preclinical rationale, said Harris. Various methods have been tested to lower APOBEC3B expression; however, a treatment has yet to be fully developed for clinical studies.

References

1. Sieuwerts AM, Willis S, Burns MB, at al. Elevated APOBEC3B Correlates with Poor Outcomes for Estrogen-Receptor-Positive Breast Cancers. Horm Cancer. 2014;5(6):405—413.
2. Sieuwerts AM, Meijer-van Gelder ME, Sweep FCGJ, et al. High APOBEC3B mRNA levels in estrogen receptor-positive primary tumors predict short time to progression for hormone-naive breast cancer patients treated with 1st line tamoxifen. Presented at: 2015 AACR Annual Meeting; April 18-22; Philadelphia, PA. Abstract LB-118.
3. Harris R, Law E, Sieuwerts AM, et al. Tamoxifen resistance driven by the DNA cytosine deaminase APOBEC3B in recurrent estrogen receptor positive breast cancer. Presented at: San Antonio Breast Cancer Symposium; December 8-12, 2015; San Antonio, TX. Abstract S4-07.

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