Daniel W. Sherbenou

Characterization of Murine JAK2V617F-Positive Myeloproliferative Disease

The JAK2(V617F) mutation is present in almost all patients with polycythemia vera (PV), large proportions of patients with essential thrombocythemia and idiopathic myelofibrosis, and less frequently in atypical myeloproliferative disorders (MPD). We show that transplantation of JAK2(V617F)-transduced bone marrow into BALB/c mice induces MPD reminiscent of human PV, characterized by erythrocytosis, granulocytosis, extramedullary hematopoiesis, and bone marrow fibrosis, but not thrombocytosis. Fluorescence-activated cell sorting of bone marrow and spleen showed proportional expansion of common myeloid progenitors, granulocyte-monocyte and megakaryocyte-erythrocyte progenitors. Megakaryocyte and late erythroid progenitors were dramatically increased, with only modest expansion of early erythroid progenitors. Erythropoietin (Epo) receptor expression was reduced on early, but normal on late erythroblasts. Serum levels of Epo and granulocyte colony-stimulating factor, but not granulocyte macrophage colony-stimulating factor, were reduced, whereas tumor necrosis factor-alpha was increased, possibly exerting a negative effect on JAK2(V617F)-negative hematopoiesis. These data suggest that erythrocytosis and granulocytosis in JAK2(V617F) mice are the net result of a complex interplay between cell intrinsic and extrinsic factors. There were no thromboembolic events and no animals succumbed to their disease, implicating additional factors in the manifestation of human disease. The disease was not transplantable and prolonged observation showed normalization of blood counts in most JAK2(V617F) mice, suggesting that the mutation may not confer self-renewal capacity.

Applying the discovery of the Philadelphia chromosome

The identification of the Philadelphia chromosome in cells from individuals with chronic myelogenous leukemia (CML) led to the recognition that the BCR-ABL tyrosine kinase causes CML. This in turn led to the development of imatinib mesylate, a clinically successful inhibitor of the BCR-ABL kinase. Incorporating the use of markers of BCR-ABL kinase inhibition into clinical trials led to the realization that imatinib-resistant kinase domain mutations are the major cause of relapse during imatinib therapy and the subsequent development of new inhibitors to treat CML patients. The development of imatinib validates an emerging paradigm in cancer, in which a tumor is defined by genetic abnormalities and effective therapies are developed that target events critical to the growth and survival of a specific tumor.

Combined Abl Inhibitor Therapy for Minimizing Drug Resistance in Chronic Myeloid Leukemia: Src/Abl Inhibitors Are Compatible with Imatinib

Purpose: Chronic myeloid leukemia (CML) is effectively treated with imatinib. However, reactivation of Bcr-Abl via kinase domain mutations that reduce sensitivity to imatinib can cause relapse. As combination therapy is frequently used to prevent emergence of resistance, the combination of imatinib with an inhibitor of imatinib-resistant Bcr-Abl mutants (e.g., Src/Abl inhibitors AP23848 and BMS-354825) was investigated. Experimental Design: To test this approach, cellular proliferation and Bcr-Abl tyrosine phosphorylation assays were done on Ba/F3 cells expressing wild-type (WT) Bcr-Abl and four common imatinib-resistant mutants (Y253F, E255K, T315I, and M351T). Colony-forming assays with primary CML cells were also done. Results: Both Src/Abl inhibitors retained full inhibitory capacity when coadministered with imatinib at concentrations above typical clinical levels. For cells expressing WT Bcr-Abl or the marginally imatinib-resistant mutant M351T, inclusion of imatinib at therapeutic levels enhanced the effects of the Src/Abl inhibitors. By comparison, for the highly imatinib-resistant mutants Y253F and E255K, inclusion of imatinib at clinical levels resulted in only a slight enhancement beyond the effects of the Src/Abl inhibitors. None of the inhibitors affected Bcr-Abl T315I cells. Colony-forming assays with primary CML cells yielded analogous results. Conclusions: Our results indicate that Src/Abl inhibitors are compatible with imatinib and suggest that combined Abl inhibitor therapy is a feasible treatment strategy for patients with CML.

Mutations of the BCR-ABL-kinase domain occur in a minority of patients with stable complete cytogenetic response to imatinib

Residual leukemia is demonstrable by reverse transcriptase-polymerase chain reaction in most patients with chronic myeloid leukemia who obtain a complete cytogenetic response (CCR) to imatinib. In patients who relapse during imatinib therapy, a high rate of mutations in the kinase domain of BCR-ABL have been identified, but the mechanisms underlying disease persistence in patients with a CCR are poorly characterized. To test whether kinase domain mutations are a common mechanism of disease persistence, we studied patients in stable CCR. Mutations were demonstrated in eight of 42 (19%) patients with successful amplification and sequencing of BCR-ABL. Mutation types were those commonly associated with acquired drug resistance. Four patients with mutations had a concomitant rise of BCR-ABL transcript levels, two of whom subsequently relapsed; the remaining four did not have an increase in transcript levels and follow-up samples, when amplifiable, were wild type. BCR-ABL-kinase domain mutations in patients with a stable CCR are infrequent, and their detection does not consistently predict relapse. Alternative mechanisms must be responsible for disease persistence in the majority of patients.

BCR-ABL SH3-SH2 domain mutations in chronic myeloid leukemia patients on imatinib

Point mutations in the kinase domain of BCR-ABL are the most common mechanism of drug resistance in chronic myeloid leukemia (CML) patients treated with ABL kinase inhibitors, including imatinib. It has also been shown in vitro that mutations outside the kinase domain in the neighboring linker, SH2, SH3, and Cap domains can confer imatinib resistance. In the context of ABL, these domains have an autoinhibitory effect on kinase activity, and mutations in this region can activate the enzyme. To determine the frequency and relevance to resistance of regulatory domain mutations in CML patients on imatinib, we screened for such mutations in a cohort of consecutive CML patients with various levels of response. Regulatory domain mutations were detected in 7 of 98 patients, whereas kinase domain mutations were detected in 29. One mutation (T212R) conferred in vitro tyrosine kinase inhibitor resistance and was associated with relapse, whereas most other mutations did not affect drug sensitivity. Mechanistic studies showed that T212R increased the activity of ABL and BCR-ABL and that T212R-induced resistance may be partially the result of stabilization of an active kinase conformation. Regulatory domain mutations are uncommon but may explain resistance in some patients without mutations in the kinase domain.

Characterization of BCR-ABL deletion mutants from patients with chronic myeloid leukemia

The BCR-ABL oncogenic tyrosine kinase causes chronic myeloid leukemia and is the target for imatinib therapy. During imatinib treatment, cells are selected in some patients with BCR-ABL kinase domain mutations that render decreased drug sensitivity. In addition, some patients express deletion mutants of BCR-ABL, apparently due to missplicing. Most commonly these deletion mutants lack a significant portion of the kinase domain that includes the P-loop. We describe a screen for such mutations in patients with CML and demonstrate that they are not oncogenic and are catalytically inactive. We hypothesized that coexpressing BCR-ABL deletion mutants has a dominant-negative effect on the native form through heterocomplex formation. However, upon coexpression of native and deletion mutant BCR-ABL in Ba/F3 cells, growth factor independence is maintained and signaling is activated normally. Despite this, these cells have increased imatinib sensitivity compared to cells expressing only native BCR-ABL. Thus, it will be important to investigate the prognostic impact of coexpression of deletion mutants in CML patients during imatinib treatment.

The development of potential antibody-based therapies for myeloma

With optimal target antigen selection antibody-based therapeutics can be very effective agents for hematologic malignancies, but none have yet been approved for myeloma. Rituximab and brentuximab vedotin are examples of success for the naked antibody and antibody-drug conjugate classes, respectively. Plasma cell myeloma is an attractive disease for antibody-based targeting due to target cell accessibility and the complementary mechanism of action with approved therapies. Initial antibodies tested in myeloma were disappointing. However, recent results from targeting well-characterized antigens have been more encouraging. In particular, the CD38 and CD138 targeted therapies are showing single-agent activity in early phase clinical trials. Here we will review the development pipeline for naked antibodies and antibody-drug conjugates for myeloma. There is clear clinical need for new treatments, as myeloma inevitably becomes refractory to standard agents. The full impact is yet to be established, but we are optimistic that the first FDA-approved antibody therapeutic(s) for this disease will emerge in the near future.

Antibody-drug conjugate targeting CD46 eliminates multiple myeloma cells

Multiple myeloma is incurable by standard approaches because of inevitable relapse and development of treatment resistance in all patients. In our prior work, we identified a panel of macropinocytosing human monoclonal antibodies against CD46, a negative regulator of the innate immune system, and constructed antibody-drug conjugates (ADCs). In this report, we show that an anti-CD46 ADC (CD46-ADC) potently inhibited proliferation in myeloma cell lines with little effect on normal cells. CD46-ADC also potently eliminated myeloma growth in orthometastatic xenograft models. In primary myeloma cells derived from bone marrow aspirates, CD46-ADC induced apoptosis and cell death, but did not affect the viability of nontumor mononuclear cells. It is of clinical interest that the CD46 gene resides on chromosome 1q, which undergoes genomic amplification in the majority of relapsed myeloma patients. We found that the cell surface expression level of CD46 was markedly higher in patient myeloma cells with 1q gain than in those with normal 1q copy number. Thus, genomic amplification of CD46 may serve as a surrogate for target amplification that could allow patient stratification for tailored CD46-targeted therapy. Overall, these findings indicate that CD46 is a promising target for antibody-based treatment of multiple myeloma, especially in patients with gain of chromosome 1q.

Cell-type specific potent Wnt signaling blockade by bispecific antibody

Cell signaling pathways are often shared between normal and diseased cells. How to achieve cell type-specific, potent inhibition of signaling pathways is a major challenge with implications for therapeutic development. Using the Wnt/β-catenin signaling pathway as a model system, we report here a novel and generally applicable method to achieve cell type-selective signaling blockade. We constructed a bispecific antibody targeting the Wnt co-receptor LRP6 (the effector antigen) and a cell type-associated antigen (the guide antigen) that provides the targeting specificity. We found that the bispecific antibody inhibits Wnt-induced reporter activities with over one hundred-fold enhancement in potency, and in a cell type-selective manner. Potency enhancement is dependent on the expression level of the guide antigen on the target cell surface and the apparent affinity of the anti-guide antibody. Both internalizing and non-internalizing guide antigens can be used, with internalizing bispecific antibody being able to block signaling by all ligands binding to the target receptor due to its removal from the cell surface. It is thus feasible to develop bispecific-based therapeutic strategies that potently and selectively inhibit signaling pathways in a cell type-selective manner, creating opportunity for therapeutic targeting.

Targeting CD46 for both adenocarcinoma and neuroendocrine prostate cancer.

Although initially responsive to androgen signaling inhibitors (ASIs), metastatic castration-resistant prostate cancer (mCRPC) inevitably develops and is incurable. In addition to adenocarcinoma (adeno), neuroendocrine prostate cancer (NEPC) emerges to confer ASI resistance. We have previously combined laser capture microdissection and phage antibody display library selection on human cancer specimens and identified novel internalizing antibodies binding to tumor cells residing in their tissue microenvironment. We identified the target antigen for one of these antibodies as CD46, a multifunctional protein that is best known for negatively regulating the innate immune system. CD46 is overexpressed in primary tumor tissue and CRPC (localized and metastatic; adeno and NEPC), but expressed at low levels on normal tissues except for placental trophoblasts and prostate epithelium. Abiraterone- and enzalutamide-treated mCRPC cells upregulate cell surface CD46 expression. Genomic analysis showed that the CD46 gene is gained in 45% abiraterone-resistant mCRPC patients. We conjugated a tubulin inhibitor to our macropinocytosing anti-CD46 antibody and showed that the resulting antibody-drug conjugate (ADC) potently and selectively kills both adeno and NEPC cell lines in vitro (sub-nM EC50) but not normal cells. CD46 ADC regressed and eliminated an mCRPC cell line xenograft in vivo in both subcutaneous and intrafemoral models. Exploratory toxicology studies of the CD46 ADC in non-human primates demonstrated an acceptable safety profile. Thus, CD46 is an excellent target for antibody-based therapy development, which has potential to be applicable to both adenocarcinoma and neuroendocrine types of mCRPC that are resistant to current treatment.