Background: Mitogen-activated protein kinase (MAPK) pathway components are frequently mutated in cancer, and abnormal MAPK signaling can drive malignancy by promoting tumor survival and proliferation. Furthermore, work in solid tumors shows that the presence of BRAFV600E further enhances tumor-induced immune suppression, but if and how this occurs in a disseminated disease such as leukemia is unknown. MAPK mutations occur in nearly 100% of classic hairy cell leukemia (HCL) and approximately 10% of chronic lymphocytic leukemia (CLL). Furthermore, up to 30% of B cell lymphomas exhibit activated MAPK signaling via expression of a BRAF pseudogene. However, the pathomechanisms of these abnormalities and the clinical utility of MAPK inhibitors in hematologic malignancies are unclear, and few models are available to evaluate this. To study these aspects, we developed in vitro and in vivo models of BRAF-mutated B cell malignancy.
Methods: To mechanistically interrogate the role of BRAFV600E in malignant B cells, we transfected the CLL cell line OSUCLL with doxycycline-inducible constructs containing normal BRAF or BRAFV600E and incubated the cells -/+ doxycycline. These cells were also co-cultured with healthy donor T cells pre-incubated with anti-CD3/anti-CD28, and T cell proliferation was measured by flow cytometry. Cytokines and surface proteins were assessed by flow cytometry. To mimic human HCL, we developed a transgenic mouse model of BRAFV600E B cell leukemia. Mice with Cre-activatable BRAFV600E expression were crossed with mice carrying Cre under the control of the CD19 promoter. CD19-Cre x BRAFV600E or CD19-Cre only mice were then crossed with the well-characterized Eµ-TCL1 model of CLL to generate mice with spontaneous B cell leukemia expressing either wild-type BRAF (CD19-Cre x TCL1) or BRAFV600E (BRAFVE x CD19-Cre x TCL1) under the native BRAF promoter. B cell restricted expression of BRAFV600E was confirmed by immunoblots of purified B and T cells using a BRAFV600E specific antibody. Blood and spleen cells were examined by flow cytometry. For adoptive transfer experiments, leukemia cells (2x10^7) from transgenic mice were introduced intravenously into syngeneic healthy adult animals.
Results: OSUCLL cells expressing BRAFV600E showed no significant changes in growth vs. wild-type BRAF expressing cells, but more strongly inhibited anti-CD3/CD28-induced proliferation of normal donor T cells. Transwell assays showed this effect was due both to soluble and contact-dependent factors. Expression of PD-L1 was not different between cells expressing normal and mutated BRAF, indicating this common checkpoint molecule is not the reason for greater immune suppression in this context. TNF levels were higher in BRAFV600E expressing cells and reduced by vemurafenib, but a TNF neutralizing antibody did not alter the inhibitory effect of BRAFV600E expressing cells on T cell proliferation. BRAFV600E x CD19-Cre x TCL1 mice developed B cell leukemia significantly earlier (median 4.9 vs. 8.1 months; P<0.001) and had significantly shorter lifespan (median 7.3 vs. 12.1 months; P<0.001) than their wild-type BRAF counterparts. In contrast to effects of BRAFV600E described in some solid tumors, BRAFV600E expression in B cells had no impact on the rate of B cell proliferation in vivo and only modestly reduced spontaneous apoptosis. To study the effects of this activating mutation on tumor-mediated immune suppression in vivo, leukemia cells from BRAFV600E x CD19-Cre x TCL1 or CD19-Cre x TCL1 mice were adoptively transferred into syngeneic mice. In 3 separate studies, tumor cells from BRAFV600E mice produced leukemia (>10% CD5+/CD19+ cells in blood) sooner than CD19-Cre x TCL1 tumor cells. In mice matched for disease load, BRAFV600E B cells produced a greater negative impact on T cells as evidenced by lower overall percentage of T cells, increased expression of T cell exhaustion markers PD-1, CD244, and CD160, and higher percentage of CD44+ memory T cells. Current studies are investigating the mechanism of these effects in vivo as well as the potential for pharmacologic reversal.
Conclusions: Together, these results demonstrate the immune-suppressive impact of BRAFV600E in B-cell leukemias and introduce a novel mouse model to develop rational combination strategies to both directly target the tumor cell and overcome tumor-mediated immune evasion.
Lozanski:Genentech: Research Funding; Stemline Therapeutics Inc.: Research Funding; Beckman Coulter: Research Funding; Boehringer Ingelheim: Research Funding.
Asterisk with author names denotes non-ASH members.