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A ROR1 small molecule inhibitor (KAN0441571C) induced significant apoptosis of ibrutinib‐resistant ROR1+ CLL cells

Abstract ROR1 – a receptor tyrosine kinase – is overexpressed in CLL. Ibrutinib, a Bruton's tyrosine kinase inhibitor, is clinically effective in CLL but patients may develop resistance. We evaluated the effect of an ROR1 inhibitor, KAN0441571C, in CLL cells from six patients obtained before and after developing resistance to ibrutinib. The ROR1 inhibitor induced apoptosis in ibrutinib‐resistant CLL cells to the same degree as in ibrutinib‐sensitive cells and dephosphorylated ROR1. This was also noted in one patient who became resistant to both ibrutinib and the Bcl‐2 inhibitor venetoclax. The combination of ROR1 inhibitor and venetoclax had a synergistic apoptotic effect on ibrutinib‐resistant cells.


INTRODUCTION
The ROR1 receptor tyrosine kinase is expressed during embryogenesis but down-regulated in the majority of normal adult tissues. However, ROR1 is expressed in various malignancies, which is of importance in the oncogenic process and of significance for cancer cell proliferation, survival and metastatic potential [1]. ROR1 expression in malignancies was initially noted in chronic lymphocytic leukemia (CLL) [2]. ROR1 could also be found in normal B-cell precursors in the bone marrow at an intermediate stage of maturation, but early and late B-cell precursors did not express ROR1 [1] suggesting that ROR1 targeted therapies may not affect normal B cells. High expression of ROR1 was related to enhanced constitutive activation of AKT and associated signaling molecules compared to cells with low ROR1 expression [3]. In CLL, high expression of activated ROR1 was associated with progressive disease [4]. An ROR1 small molecule inhibitor (SMI) could induce dephosphorylation of ROR1 in CLL cells as well as of canonical and non-canonical Wnt signaling proteins with subsequent induction of apoptosis [5]. ROR1 has been shown to crosstalk with the B-cell receptor (BCR) through the BCR complex and Bruton's tyrosine kinase (BTK) as well as with associated signaling molecules in acute lymphoblastic leukemia (ALL) B cells [3]. BTK is an important tyrosine kinase in the BCR signaling pathway driving the BCR signaling cascade leading to activation of downstream NF-κB and phosphatidylinositol-3-kinase (PI3K) with a pro-survival effect of CLL clone [6].
Ibrutinib is a covalent, irreversible SMI binding to BTK and highly effective for the treatment of CLL even in unselected (real-world) patients [6]. The mechanism of action (MOA) is partly due to the ability to inhibit signal transduction through the BCR pathway. Even though most CLL patients initially respond to ibrutinib, an increasing number of patients develop resistance to the drug which is associated with mutation in the BTK gene at the ibrutinib binding site as C481S as well as mutations in the immediate downstream effector phospholipase Cγ2 (PLCγ2) (R665W and L845F) domain [7]. These mutations are detected in about 80% of CLL patients developing resistance to ibrutinib [8]. Thus, there is a great medical need to develop drugs with other MOA than ibrutinib. Venetoclax (a Bcl-2 inhibitor) has been shown to be effective in ibrutinib-resistant patients [9]. We have previously shown in preclinical models that small molecules inhibiting ROR1, KAN0439834 and KAN0441571C, induced tumor cell death in CLL and DLBCL cells which express activated ROR1 [5,10].
In this study, tumor cell death in vitro after incubation with an ROR1 inhibitor (KAN0441571C) was compared in ROR1 + CLL cells obtained from patients before and after developing clinical resistance to ibrutinib. Venetoclax was used as a control as this drug may be effective in ibrutinib-resistant cases. We also evaluated the combination of venetoclax and ROR1 inhibitor for induction of apoptosis in ibrutinib-resistant cells. Moreover, a patient who had developed double-refractoriness to both ibrutinib and venetoclax was also included.

RESULTS AND DISCUSSION
KAN0441571C induced significant apoptosis to the same extent in both ibrutinib-sensitive (BTK unmutated) and ibrutinib-resistant (BTK-mutated) ROR1 + CLL cells from the same patient. Representative dose-response curves of a patient including EC 50 values are shown in Figure 1A. The slope of the dose-response curve was as expected for a drug with a pro-apoptotic MOA. The apoptotic effect of venetoclax in the same patient is shown in Figure 1B. EC 50 values for apoptosis of ibrutinib-sensitive and -resistant CLL cells, respectively, incubated with KAN0441571C for all the individual patients are depicted in Figure 1C. There was no statistically significant difference between ibrutinib-sensitive and -resistant cells in response to ROR1 inhibition.
This was also noted for venetoclax treatment in vitro ( Figure 1D) (Table   S1). In ibrutinib-resistant cases, there was a trend (p = 0.09) towards a high EC 50 conc. of the ROR1 inhibitor for induction of apoptosis with increasing frequency of BTK-mutated cells ( Figure S1). No such correlation for the EC 50 conc. of venetoclax was noted (data not shown).
One patient developed first clinical resistance to ibrutinib and then also to venetoclax. As can be seen in Figure 1E, the apoptotic response to the ROR1 inhibitor was similar in ibrutinib-sensitive and -resistant cells as well as in double-refractory cells. However, ibrutinib-sensitive and -resistant cells responded equally well to venetoclax while doublerefractory cells required 10-15 times higher concentration of venetoclax to achieve the same apoptotic effect as compared to that required for ibrutinib-sensitive and -resistant cells ( Figure 1F).
Ibrutinib has been shown to have limited direct pro-apoptotic activity in vitro and requires interfering with cross-talk between CLL cells and the lymph node microenvironment. Advanced ex vivo drug testing systems are necessary [12]. Thus, the present ex vivo model does not allow evaluation of the apoptotic effect of ibrutinib.
KAN0441571C consistently dephosphorylated ROR1 in both ibrutinib-sensitive and -resistant cells (Figure 2A) while the effect on BTK phosphorylation was varying but overall there was a statistically significant increase ( Figure 2B). There was no significant difference between the effect on ibrutinib-sensitive and -resistant cells for either ROR1 or BTK phosphorylation.
The combination of KAN0441571C and venetoclax showed a synergistic apoptotic effect in ibrutinib-resistant CLL cells in all the six patients ( Figure S2).
Crosstalk between the BCR complex and ROR1 signaling through activation of BTK, has been described in CLL, ALL, Burkitt's lymphoma and mantle cell lymphoma (MCL) [3,13,14]. In ALL cells, antagonize ROR1 mediated apoptotic response [14]. The results are in line with our finding of a synergistic apoptotic effect in CLL cells by targeting both ROR1 and Bcl-2 in ibrutinib-resistant cells. We have also previously shown that the combination of ibrutinib and an ROR1 SMI (KAN0439834) augmented the killing of pancreatic cancer cells compared to either drug alone [15].
There is still a great medical need for novel drugs to be developed in CLL and also to be tested in combinations. Our results indicate that apoptosis can be induced by a ROR1 inhibitor in ibrutinib-resistant CLL cells and preliminary also in venetoclaxresistant cells. Furthermore, combination of ROR1 targeting drugs with ibrutinib or venetoclax seems to increase the tumor cell killing in vitro.

ACKNOWLEDGEMENT
This study was supported by grants from the Swedish Cancer Society, the Cancer Society in Stockholm, Åke Olsson Foundation, Region Stockholm and Cancer and Allergy Foundation. For skillfull secreterial assistance, we thank Mrs Leila Relander.