Shih-Shih Chen

Leukemia cell proliferation and death in chronic lymphocytic leukemia patients on therapy with the BTK inhibitor ibrutinib

BACKGROUND. Ibrutinib is an effective targeted therapy for patients with chronic lymphocytic leukemia (CLL) that inhibits Brutons tyrosine kinase (BTK), a kinase involved in B cell receptor signaling. METHODS. We used stable isotopic labeling with deuterated water (2H2O) to measure directly the effects of ibrutinib on leukemia cell proliferation and death in 30 patients with CLL. RESULTS. The measured average CLL cell proliferation (birth) rate before ibrutinib therapy was 0.39% of the clone per day (range 0.17%-1.04%); this decreased to 0.05% per day (range 0%-0.36%) with treatment. Death rates of blood CLL cells increased from 0.18% per day (average, range 0%-0.7%) prior to treatment to 1.5% per day (range 0%-3.0%) during ibrutinib therapy, and they were even higher in tissue compartments. CONCLUSIONS. This study provides the first direct in vivo measurements to our knowledge of ibrutinibs antileukemia actions, demonstrating profound and immediate inhibition of CLL cell proliferation and promotion of high rates of CLL cell death. TRIAL REGISTRATION. This trial was registered at clinicaltrials.gov (NCT01752426). FUNDING. This study was supported by a Cancer Center Support Grant (National Cancer Institute grant P30 CA016672), an NIH grant (CA081554) from the National Cancer Institute, MD Andersons Moon Shots Program in CLL, and Pharmacyclics, an AbbVie company.

Murine Genetically Engineered and Human Xenograft Models of Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is a genetically complex disease, with multiple factors having an impact on onset, progression, and response to therapy. Genetic differences/abnormalities have been found in hematopoietic stem cells from patients, as well as in B lymphocytes of individuals with monoclonal B-cell lymphocytosis who may develop the disease. Furthermore, after the onset of CLL, additional genetic alterations occur over time, often causing disease worsening and altering patient outcomes. Therefore, being able to genetically engineer mouse models that mimic CLL or at least certain aspects of the disease will help us understand disease mechanisms and improve treatments. This notwithstanding, because neither the genetic aberrations responsible for leukemogenesis and progression nor the promoting factors that support these are likely identical in character or influences for all patients, genetically engineered mouse models will only completely mimic CLL when all of these factors are precisely defined. In addition, multiple genetically engineered models may be required because of the heterogeneity in susceptibility genes among patients that can have an effect on genetic and environmental characteristics influencing disease development and outcome. For these reasons, we review the major murine genetically engineered and human xenograft models in use at the present time, aiming to report the advantages and disadvantages of each.

Adenosine signaling mediates hypoxic responses in the chronic lymphocytic leukemia microenvironment

The chronic lymphocytic leukemia (CLL) niche is a closed environment where leukemic cells derive growth and survival signals through their interaction with macrophages and T lymphocytes. Here, we show that the CLL lymph node niche is characterized by overexpression and activation of HIF-1α, which increases adenosine generation and signaling, affecting tumor and host cellular responses. Hypoxia in CLL lymphocytes modifies central metabolic pathways, protects against drug-driven apoptosis, and induces interleukin 10 (IL-10) production. In myeloid cells, it forces monocyte differentiation to macrophages expressing IRF4, IDO, CD163, and CD206, hallmarks of the M2 phenotype, which promotes tumor progression. It also induces IL-6 production and enhances nurturing properties. Low oxygen levels decrease T-cell proliferation, promote glycolysis, and cause the appearance of a population of PD-1+ and IL-10-secreting T cells. Blockade of the A2A adenosine receptor counteracts these effects on all cell populations, making leukemic cells more susceptible to pharmacological agents while restoring immune competence and T-cell proliferation. Together, these results indicate that adenosine signaling through the A2A receptor mediates part of the effects of hypoxia. They also suggest that therapeutic strategies to inhibit the adenosinergic axis may be useful adjuncts to chemotherapy or tyrosine kinase inhibitors in the treatment of CLL patients.

Combined BTK and PI3Kδ Inhibition with Acalabrutinib and ACP-319 Improves Survival and Tumor Control in CLL Mouse Model

Purpose: Targeting the B-cell receptor (BCR) pathway with inhibitors of Bruton tyrosine kinase (BTK) and PI3Kδ is highly effective for the treatment of chronic lymphocytic leukemia (CLL). However, deep remissions are uncommon, and drug resistance with single-agent therapy can occur. In vitro studies support the effectiveness of combing PI3Kδ and BTK inhibitors. Experimental Design: As CLL proliferation and survival depends on the microenvironment, we used murine models to assess the efficacy of the BTK inhibitor acalabrutinib combined with the PI3Kδ inhibitor ACP-319 in vivo. We compared single-agent with combination therapy in TCL1-192 cell–injected mice, a model of aggressive CLL. Results: We found significantly larger reductions in tumor burden in the peripheral blood and spleen of combination-treated mice. Although single-agent therapy improved survival compared with control mice by a few days, combination therapy extended survival by over 2 weeks compared with either single agent. The combination reduced tumor proliferation, NF-κB signaling, and expression of BCL-xL and MCL-1 more potently than single-agent therapy. Conclusions: The combination of acalabrutinib and ACP-319 was superior to single-agent treatment in a murine CLL model, warranting further investigation of this combination in clinical studies. Clin Cancer Res; 23(19); 5814–23. ©2017 AACR.

An IgG1-like bispecific antibody targeting CD52 and CD20 for the treatment of B-cell malignancies

Bispecific antibodies (biAb) targeting two different antigens or two distinct epitopes on the same antigen have demonstrated broad therapeutic utility. CD52 and CD20 are co-expressed on the cell surface of malignant B cells in B-cell non-Hodgkin lymphoma (B-NHL) and chronic lymphocytic leukemia (CLL) and increased expression of both antigens is detected on dividing or recently divided cells (proliferative fraction) in CLL. The CD52-targeting monoclonal antibody (mAb) alemtuzumab (atz) not only depletes malignant B cells but also healthy CD52+ B and T lymphocytes and monocytes, causing severe immunosuppression. Loss of CD20 can occur in CLL after treatment with rituximab (rtx) and other CD20-targeting mAbs. To broaden the benefit of atz and rtx, we engineered an IgG1-like biAb, atzu202f×u202frtx scFv-Fc. The Fc fragment of the biAb facilitates purification by Protein A affinity chromatography and supports a longer circulatory half-life. While atzu202f×u202frtx scFv-Fc retained both antigen binding specificities, it showed superior binding to CD52+CD20+ B cells compared to CD52+CD20- T cells. Moreover, atzu202f×u202frtx scFv-Fc mediated potent complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) in vitro and exhibited B-cell depleting but T-cell sparing activities in vivo in a CLL patient-derived xenograft model. B-cell depletion was more pronounced for cells of the proliferative fraction.

Abstract 960: Mechanisms underlying primary ibrutinib sensitivity in CLL

The Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib is efficacious in B cell malignancies including chronic lymphocytic leukemia (CLL). Although the majority of CLL patients respond to ibrutinib, CLL clones with unmutated IGHV gene (U-CLL) are significantly more sensitive to ibrutinib than clones with mutated IGHV (M-CLL). About 77% of U-CLL but only 33% M-CLL patients experience a partial or complete response. Since ibrutinib resistance occurs, understanding the molecular mechanisms underlying this difference will improve clinical practice. Here we hypothesized that the difference in ibrutinib sensitivity between U-CLL and M-CLL was due to a greater vulnerability of U-CLL B cells to the loss of environmental prosurvival signals. We first evaluated 28 treatment naive CLL cases receiving ibrutinib. We defined not only the characteristic CLL B cell but also T cell lymphocytosis and the latter resolved faster in patients with good prognosis. After 1 treatment cycle, CLL T cells failed to proliferate upon mitogenic stimulation and to home to solid tissues after transfer into NSG mice. Without BCR signaling and T cell support, the CLL B cell proliferative fraction was diminished and the remaining B cells had elevated but dysfunctional surface membrane (sm) CXCR4 based on impaired recycling, internalization and signaling. Ibrutinib also inhibited CXCR4 phosphorylation at ser324/325/339 at a greater level in U-CLL. We then investigated ibrutinib mediated changes in microenvironment in a xenograft mouse model using primary cells from 3 U-CLL and 3 M-CLL cases. Ibrutinib significantly inhibited CLL cell growth in spleen; the treated CLL cells developed elevated but dysfunctional smCXCR4. The inhibition on tumor growth and impaired CXCR4 occurred to much greater extent in U-CLL. Notably, ibrutinib also significantly inhibited T cell growth only in U-CLL cases. Thus, ibrutinib inhibited CXCR4 signaling and CLL B-T cell crosstalk essential for tumor growth in tissue niches, especially in U-CLL cases that has limited B-lymphocytosis. Finally, molecules controlling the vulnerability of U-CLL B cells to death in the absence of environmental prosurvival signals were examined in 15 patients. Ibrutinib reduced BCL2 protein levels in U-CLL but not M-CLL; levels of BCL-XL and MCL-1 were unchanged. Consistent with these results, U-CLL but not M-CLL cell survival in vitro was promoted by T cell stimulation that led to upregulation of prosurvival BCL2. These findings suggest enhanced CLL B cell death by inhibiting BCL2 in U-CLL after the loss of environmental prosurvival signals. Altogether, ibrutinib inhibits CLL-microenvironment crosstalk by blocking CXCR4 signaling and T cell support. U-CLL cells had a greater vulnerability to the loss of environmental prosurvival signals after ibrutinib inhibition. Our data demonstrate mechanisms underlying the differences in primary ibrutinib sensitivity between U- and M- CLL patients, supporting the use of inhibitors of BTK and BCL2 in CLL. Note: This abstract was not presented at the meeting. Citation Format: Shih-Shih Chen, Priyadarshini Ravichandran1, Yasmine Kieso, Jacqueline C. Barrientos, Jan Burger, Kanti R. Rai, Nicholas Chiorazzi. Mechanisms underlying primary ibrutinib sensitivity in CLL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 960. doi:10.1158/1538-7445.AM2017-960