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Harnessing negative B-cell selection to overcome drug-resistance in B-cell malignancies

Figure 1: Leveraging mechanisms of negative B-cell selection to overcome drug-resistance.

Thresholds of B-cell selection based on signaling strength of the BCR (left) or an oncogenic mimic (BCR-ABL1; right) are depicted. BCR-signals are amplified by kinases (e.g. SYK, PI3K) and attenuated by inhibitory phosphatases (e.g. SHIP1, PTEN) to achieve intermediate signaling strength (middle).
When B-cells lack BCR-signaling (Anergic; bottom left) or in the presence of kinase-inhibitor treatment in B-cell malignancies (bottom right), signaling strength falls below minimum levels for proliferation and survival.
Conversely, upon BCR-hyperactivation in autoreactive B-cells (top left) or kinase-hyperactivation in B-cell malignancies (e.g. by kinase-superagonists or small-molecule phosphatase-inhibitors, top right), signaling strength exceeds maximum thresholds and triggers negative selection and cell death.

To prevent the production of harmful autoantibodies and autoimmune disease, autoreactive B-cells are eliminated by a process we termed negative selection. Contrary to established dogma, these mechanisms are not only active in preventing autoimmune disease but as demonstrated by our work (Trageser et al., 2009; Swaminathan et al., 2013; Chen et al., 2015; Shojaee et al., 2016), they also represent an entirely novel class of therapeutic targets in B-cell tumors, including B-cell acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL).

Despite oncogenic transformation, these B-cell malignancies remain fully sensitive to mechanisms of negative selection. Targeted hyperactivation of B-cell receptor (BCR)-downstream kinases mimics excessive signaling-strength from an autoreactive BCR and triggers negative selection. This can be achieved by pharmacological kinase hyperactivation using superagonists of SYK and other BCR-proximal kinases (Figure 1). Targeted cancer-therapy is traditionally focused on kinase-inhibitors to suppress oncogenic signaling. Their concept of targeted kinase-hyperactivation effectively represents the opposite (Müschen, 2018; Müschen, 2019)

Feedback control of oncogenic signaling in B-cell malignancies as a therapeutic target

Unlike other cell-types, B-cells rely on a ‘Goldilocks’ principle that selects B-cell clones for survival and proliferation based on intermediate B-cell receptor (BCR) signaling strength. In analogy to normal B-cells, we discovered that in many B-cell malignancies, the transforming oncogene mimics a constitutively active (pre-)BCR (Feldhahn et al., 2005).

Based on these observations, we demonstrated that B-cell acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) cells critically depend on robust feedback mechanisms to balance fluctuations of oncogenic BCR-signaling strength (Buchner et al., 2015: Shojaee et al., 2015; Chan et al., 2020; Lee et al., 2020). The efforts for preclinical development of this concept focused on small-molecule inhibition of DUSP6, a central feedback regulator of ERK-signaling.

Metabolic gatekeeper function of B-cell transcription factors

While B-cell transcription factors PAX5 and IKZF1 are known for their role in B-cell differentiation, we recently discovered a novel B-lymphoid transcriptional program to restrict energy abundance (Chan et al., 2017), a mechanism named ‘metabolic gatekeeper’ (Müschen, 2019). Oncogenic signaling and increased proliferation results in higher energy demands of transformed B-cells than their normal counterparts.

Hence, B-cell transcription factors function as metabolic gatekeepers (Chan et al., 2017; Schjerven et al., 2017; Xiao et al., 2018; Sadras et al., 2021; Pan et al., 2021) by limiting energy-supply and safeguarding against malignant transformation by setting low thresholds for the elimination of premalignant clones based on energy-stress.

BCL6 enables stemness and a novel form of drug-resistance in B-cell leukemia

While B-cell lymphoma 6 (BCL6) is widely known as oncogene in mature B-cell lymphomas, we discovered that B-cell acute lymphoblastic leukemia (B-ALL) cells respond to treatment with tyrosine kinase inhibitors (TKI) by over 100-fold increases of BCL6-expression, which drives a previously unrecognized form of drug-resistance (Duy et al., 2011; Nahar et al., 2011; Hurtz et al., 2011).

For mechanistic studies, we developed genetic mouse models for conditional ablation of Bcl6 and a Bcl6-reporter allele (Bcl6tm1Mamu) and found that BCL6 mediates drug-resistance by induction of a quiescent stem cell-like state in B-ALL and other leukemias. Our work demonstrated that BCL6 peptide- and small-molecule antagonists restored responses to standard chemotherapy and TKI in refractory leukemias (Hurtz et al., 2019).

Immunoglobulin diversifiers in clonal evolution of B-cell precursors towards B-ALL and lymphoma

Unlike other cell-types, B-cells undergo multiple rounds of gene-recombination (mediated by the RAG1 and RAG2 enzymes) and hypermutation (mediated by AID) to evolve high-affinity antibodies. High frequencies of DNA-strand breaks engender an approximately 300-fold increased risk of malignant transformation. While AID and RAG1/2 are strictly segregated during normal B-cell development, we found that their aberrant co-expression plays a central role in clonal evolution towards leukemia (Feldhahn et al., 2007; Tsai et al., 2008; Klemm et al., 2009; Kharabi Masouleh et al., 2014; Swaminathan et al., 2015; Huang et al., 2019 ).

In addition, we demonstrated that AID enables clonal evolution towards B-cell acute lymphoblastic leukemia (B-ALL) and mantle cell lymphoma (MCL) relapse and drug-resistance (Klemm et al., 2009).