Preclinical evaluation of the Hsp90 inhibitor SNX-5422 in ibrutinib resistant CLL

<jats:title>Abstract</jats:title><jats:p>B-cell receptor (BCR) antagonists such as the BTK inhibitor ibrutinib have proven to effectively target chronic lymphocytic leukemia (CLL) tumor cells, leading to impressive response rates in these patients. However patients do still relapse on ibrutinib, and the progressive disease is often quite aggressive requiring immediate treatment. Several strategies are being pursued to treat patients who relapse on ibrutinib therapy. As the most common form of relapse is the development of a mutant form of BTK which limits ibrutinib binding, agents which lead to degradation of the BTK protein are a promising strategy. Our study explores the efficacy of the Hsp90 inhibitor, SNX-5422, in CLL. The SNX Hsp90 inhibitor was effective in primary CLL cells, as well as B-cell lines expressing either BTK wild type or C481 mutant BTK, which has been identified as the primary resistance mechanism to ibrutinib in CLL patients. Furthermore the combination of SNX-5422 and ibrutinib provided a remarkable in vivo survival benefit in the Eμ-TCL1 mouse model of CLL compared to the vehicle or single agent groups (51 day median survival in the vehicle and ibrutinib groups versus 100 day median survival in the combination). We report here preclinical data suggesting that the Hsp90 inhibitor SNX-5422, which has been pursued in clinical trials in both solid tumor and hematological malignancies, is a potential therapy for ibrutinib resistant CLL.</jats:p>

section_content: To the editor,

In the front-line setting, agents targeting B-cell receptor (BCR) signaling have shown extremely promising results, and are an option for initial therapy in patients with Chronic Lymphocytic Leukemia (CLL) [1]. For patients with relapsed CLL the median progression free survival in CLL patients treated with ibrutinib was 44. 1 months, with a cumulative overall response rate of 91%. Nevertheless, ibrutinib was discontinued in 37% of CLL patients due to progressive disease [2], and patients who relapse on ibrutinib progress quickly and have poor overall survival [3]. Patients who progress on BTK inhibitors which rely on the C481 binding site develop frequent mutations in BTK (C481S) which circumvents BTK inhibition in ~ 85% of cases [4]. Therefore combinatorial approaches to target mutant BTK could eliminate the mutant clone allowing the patient to continue on ibrutinib. 

One promising clinical strategy in patients with resistant CLL is Hsp90 inhibition to target the BTK protein. Esanex Pharmaceuticals developed a novel Hsp90 inhibitor, SNX-5422 (the prodrug of SNX-2112) which has been safely tested in multiple phase I studies in solid tumors and hematological malignancies [5] [6] [7]. In treatment-naïve primary CLL cells we see reduced proliferation with as low as 0. 1uM SNX-2112 (Fig. 1a ) including CpG stimulated primary CLL cells which mimics the natural stimulation of the tumor microenvironment (Fig. 1b ). Furthermore we found that downstream mediators of BCR signaling, BTK and AKT, are consistently down-regulated in all patient samples examined (Fig. 1c ). Furthermore while ibrutinib is able to reduce BTK autophosphorylation at Y223 in cells expressing wild type BTK protein, cells expressing C481S mutated BTK [8, 9] are resistant (Fig. 1d ). However we see a reduction in both phospho and total BTK with 0. 1uM SNX-2112 in both WT and C481S BTK cell lines. 

Next, we tested the efficacy of SNX-5422 in combination with ibrutinib using a Eμ-TCL1 CLL ibrutinib resistant model previously reported by our group [10, 11]. Mice dosed with vehicle or ibrutinib had similar overall survival, however the combination of SNX-5422 and ibrutinib provided remarkable survival benefit (Fig. 2a ; 51 day median survival in the vehicle and ibrutinib groups versus 100 day median survival in the combination; p < 0. 01). Mice treated with SNX-5422 had smaller spleens when compared to vehicle and ibrutinib treated mice (Fig. 2b, SNX-5422 + ibrutinib vs. vehicle or ibrutinib: p < 0. 001), with a trend towards decreased peripheral blood tumor development (green line, Fig. 2c ). Finally, histopathological analysis of leukemic infiltration into surrounding tissues revealed that mice treated with combination therapy had reduced severity of neoplastic infiltrates in liver, spleen, and lung (Fig. 2d ). We performed an additional in vivo study using the Eμ-BRD2 model which develops an aggressive lymphoma with splenomegaly, We did note reduced survival in the single agent SNX-5422 treated mice despite the reduced tumor burden, therefore we performed detailed histopathology in both in vivo models. Surprisingly, there was marked ulceration of the non-glandular stomach (Additional file 2; indicated by black arrows) accompanied by immune cell infiltration (indicated by red arrows), and hyperplasia of the surrounding squamous gastric mucosa (indicated by green arrows) which was less severe in mice treated with the combination. As these ulcers only occurred in the non-glandular stomach (an anatomic counterpart lacking in humans), this toxicity is unlikely to occur in human patients receiving SNX-5422, which was safe and tolerable with a recommended dose of 100 mg/m 2 QOD 3 weeks on and 1 week off in phase I solid tumor trials [5]. Interestingly, in both models these symptoms appear to be less severe in mice treated with the combination, suggesting that ibrutinib is ameliorating some of the toxicities related to SNX-5422. 

Altogether, our data suggest that Hsp90 inhibition in combination with ibrutinib may be an option for initial treatment in CLL to prevent the outgrowth of a resistant clone in patients who display high risk features that However, studies to determine the efficacy in CLL in patients receiving ibrutinib therapy (NCT02973399, NCT02914327) have not been completed due to low enrollment and have not been published. This is likely attributed to the overall success of ibrutinib and other BTK inhibitors, as well as newer generation BTK inhibitors that do not rely on the C481S binding site currently in development. Nevertheless our work shows that alternative strategies that target BTK for degradation are a promising option in BTK inhibitor resistant CLL.

section_name: Acknowledgements

section_content: Not applicable
section_name: Acknowledgements

section_name: Funding

section_content: This work was supported by the National Cancer Institute ( R35 CA197734-01, P01 CA81534, K12 CA133250, and OSUCCC Core Facilities P30 CA016058 ), Sullivan Foundation, The Leukemia and Lymphoma Society ( special scholar Grant to EH) and The D. Warren Brown Foundation.
section_name: Funding

section_name: Availability of data and materials

section_content: All data collected during this study are included in this published article or the supplementary information.
section_name: Availability of data and materials

section_name: Supplementary Information

section_content: The online version contains supplementary material available at https ://doi. org/10. 1186/s1304 5-021-01039 -9. Abbreviations CLL: Chronic lymphocytic leukemia; BCR: B-cell receptor; BTK: Bruton's tyrosine kinase; XLA: X-linked agammaglobulinemia.
section_name: Supplementary Information

section_content: TLC designed and conducted experiments, generated data and figures, analyzed data, interpreted results, and wrote the manuscript; BH conducted experiments and generated figures; AP, JT, RW and SS performed animal experiments; AL conducted all statistical analysis; EO contributed critical reagents, LA RAB, JCB and JAW planned the project, interpreted results, reviewed the manuscript; and obtained funding and EH planned the project, supervised the study, analyzed data, interpreted results, reviewed the manuscript; and obtained funding. All authors read and approved the final version of the manuscript. All authors read and approved the final manuscript.
section_name: Authors' contributions

section_content: The authors are grateful to the patients who generously provided blood for these studies. Blood from CLL patients was obtained from the Ohio State University Leukemia Tissue Bank after obtaining informed consent approved by the cancer institution review board (IRB). All mouse protocols were reviewed and approved by The Ohio State University Institutional Animal Care and Use Committee (IACUC).
section_name: Ethics approval and consent to participate

section_content: Not applicable.
section_name: Consent for publication

section_content: At the time these studies were completed, E. O held the position of chief scientific officer for Esanex who provided SNX-2112 and SNX-5422 for this work. 

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