Small Molecule Inhibitors of HER2 Inhibit Proliferative Signaling and Promote Apoptosis in Ph+ ALL
Authors: Irwin Mary E , Nelson Laura , Santiago-O'Farrill Janice M , Miller Claudia P , Siwak Doris R. , Mills Gordon B. , Li Shulin , Hughes Dennis P , Chandra Joya 
Published: October 2019

Abstract
Abstract 1397
The ERBB family of receptor tyrosine kinases (EGFR, Her-2, Her-3 and Her-4) are receptor tyrosine kinases that, through mutation or aberrant expression, serve as oncogenes by promoting hallmark behaviors of cancer in many solid tumors. Previous work has suggested that HER2 is expressed in as much as 30% of B-ALL patients, and correlates with chemoresistance. We therefore hypothesized that HER2 signaling in Ph+ ALL may augment growth signaling and promote other malignant behaviors, such as resistance to cell death and independence from growth factors. Western blot and flow cytometric analyses of two human Ph+ ALL cell lines, Z119 and Z181, revealed cell surface expression of HER2, but not other family members. To determine the role of HER2 signaling in Ph+ ALL cell lines, the pan-HER family small molecule kinase inhibitor canertinib was used, and reverse phase protein array (RPPA) was conducted in Z119 and Z181 cell lines. Briefly, lysates from canertinib treated cells were spotted using a GeneTAC™ G3 arrayer onto nitrocellulose-coated FAST® slides. Incubation of the slides was performed with forty-three antibodies directed towards various cell signaling proteins followed by colorimetric detection and results were subsequently validated by western blotting. RPPA analyses revealed that treatment with canertinib effectively diminished HER2 phosphorylation in both cell lines. Additionally, we found decreased phosphorylation of the pro-survival molecules ribosomal protein S6, p70S6kinase, and c-Src, as well as increased expression of the pro-apoptotic molecules BIM and cleaved-PARP in both Ph+ ALL cell lines. Congruent with these findings, elevated activity of the executioner caspase 3 and increased DNA fragmentation, two distinct biochemical markers of apoptosis, were present after canertinib treatment in Z181 and Z119 cells, suggesting that inhibition of HER2 signaling results in programmed cell death of Ph+ ALL cell lines. This induction of apoptosis paralleled a decrease in overall proliferation of these cell lines, further implicating HER2 signaling in proliferation of Ph+ ALL. Next, we analyzed if clinically approved inhibitors of HER2 function could be utilized to produce the same biological consequence as canertinib in Ph+ ALL cell lines. Lapatinib (Tykerb) is a dual EGFR/HER2 small molecule kinase inhibitor approved by the FDA for the treatment of breast cancer. Consistent with our results utilizing canertinib, lapatinib was capable of inhibiting proliferation of both Z119 and Z181 cell lines. Interestingly, the FDA approved monoclonal antibody HER2 inhibitor trastuzumab (Herceptin) did not inhibit proliferation of these cell lines. Similarly, trimerized herceptin conjugates, which improve internalization of HER2 receptor, also had no effect on Ph+ ALL cell line proliferation. These results highlight an important distinction between the effects of the intracellular small molecule inhibitors of HER2 and monoclonal HER2 antibodies. In particular, extracellular engagement of the HER2 receptor by monoclonal antibodies may not be effective in targeting the HER2 signaling pathways required for proliferation and survival of Ph+ ALL. Taken together, our studies suggest that HER2 may play an important role in growth and survival signaling of Ph+ ALL cell lines and inhibition of HER2 with small molecule kinase inhibitors may improve treatment regimens. Thus, additional studies are warranted to determine the importance of HER2 in clinical specimens and the potential benefit of combining HER2 inhibitor therapy with imatinib treatment for Ph+ ALL.

Disclosures:
Mills: Glaxosmithkline: Research Funding; Pfizer: Research Funding.