 2014 Genentech USA, Inc. All rights reserved. 1 Disclosure/Disclaimer The Molecular Basis of Cancer Educational Series is not intended to promote any cancer agent or class approved by the FDA or currently under clinical development. The contents of these slide presentations are owned solely by Genentech; any unauthorized uses are prohibited. These programs are intended to provide general information about the molecular basis of cancer, not medical advice for any particular patient. The information is presented on behalf of Genentech, and is consistent with FDA guidelines. BIO

 2014 Genentech USA, Inc. All rights reserved. 2 The physiologic germinal center microenvironment Ig=immunoglobulin; V-region=variable region; BCR=B-cell receptor; FDC=follicular dendritic cell. Reprinted with permission from Macmillan Publishers Ltd: Nat Rev Cancer, 2005; , copyright Küppers R. Nat Rev Cancer. 2005;5: Mantle zone Light zoneDark zone Somatic hypermutationClass switching Clonal expansion Selection Differentiation FDC T cell Mutations that increase antigen affinity Mutations that reduce antigen affinity Plasma cell Memory B cell Apoptosis No BCR Naïve B cell B-cell precursor lg gene rearrangements V-region gene recombination  As mentioned earlier, B cells are remarkable in that they can somatically alter their genomes. In addition to V(D)J rearrangement in the bone marrow, B cells undergo further antigen-driven diversification of their genomes in the GC reaction via 2 mechanisms: SHM and CSR 1 – Expansive proliferation takes place in the GC dark zone and involves SHM. SHM introduces mutations at a very high rate into the Ig-variable region genes with the activity of activation-induced deaminase (AID) 2 Mutated B cells then migrate to the GC light zone, which is rich in CD4+ T helper (T H ) cells and FDCs. GCB cells that have acquired mutations that increase BCR affinity for antigen are selected, whereas B cells with lowered BCR affinity for antigen undergo apoptosis 2 SHM has also been found to target nonimmunoglobulin genes such as Bcl-6, though the functional advantage of this is not yet clear 3 – Many GCB cells also undergo Ig class switching, allowing antigen-specific antibodies to change their effector function without changing specificity 3 CSR-inducing signals are transmitted to B cells by GC T cells that are present in the light zone 3 – Finally, GCB cells that are positively selected become either memory B cells or plasma cells and then leave the GC 2 References: 1.Lenz G, Staudt LM. Aggressive lymphomas. N Engl J Med. 2010;362: Zenz T, Mertens D, Küppers R, Döhner H, Stilgenbauer S. From pathogenesis to treatment of chronic lymphocytic leukaemia. Nat Rev Cancer. 2010;10: Klein U, Dalla-Favera R. Germinal centres: role in B ‑ cell physiology and malignancy. Nat Rev Immunol. 2008;8: Notes

3  2014 Genentech USA, Inc. All rights reserved. DAGIP 3 Ras Raf MEK MAPK MALT1Bcl-10 CARD11 BCR signaling is central to the pathogenesis of lymphoma SFK Syk NFκB PDK1 Akt PI3K Davis RE, et al. Nature. 2010;463: Staudt LM. Cold Spring Harb Perspect Biol. 2010;2:a Bcl-10=B-cell lymphoma 10; BCR=B-cell receptor; BLNK=B-cell linker; BTK=Bruton’s tyrosine kinase; CARD11=caspase recruitment domain-containing protein 11; CBM=CARMA1-Bcl10-MALT1 complex; DAG=diacylglycerol; IP3=inositol triphosphate; MALT1=mucosa-associated lymphoid tissue lymphoma translocation protein 1; MAPK=mitogen-activated protein kinase; MEK=mitogen-activated protein kinase kinase; mTOR=mammalian target of rapamycin; NFκB=nuclear factor kappa–light-chain enhancer of activated B cells; PDK1=phosphoinositide-dependent kinase-1; PI3K=phosphatidylinositol 3-kinase; PKC=protein kinase C; PLCγ2=phospholipase Cγ2; Ras=rat sarcoma; Raf=rapidly accelerating fibrosarcoma; SFK=Src family kinase; Syk=spleen tyrosine kinase. BLNK PKC   Ca 2+ PLC  2 CBM complex CD 19 BTK mTOR Antigen B-cell receptor CD79aCD79b  Proliferation  Cell survival  Proliferation 3  Most B-cell lymphomas maintain BCR expression on the cell surface, an observation that suggests malignant B cells benefit from proliferation and survival signals mediated by the BCR  Signaling through the B-cell receptor is central to the development and maintenance of normal B cells  Every normal B cell has a unique membrane-bound antibody known as the BCR that allows the cell to bind diverse antigens in the extracellular environment  This antibody portion of the BCR is coupled with a heterodimer of CD79a and CD79b. Antigen-induced aggregation of the BCR leads to phosphorylation of CD79a and CD79b tyrosine residues by the Src-family kinases, including Lyn, Fyn, and Blk. The tyrosine kinase Syk is then recruited to phosphorylated CD79a and CD79b through its tandem Src homology 2 (SH2) domains. Syk recruits a complex of Cbl-interacting protein and B-cell linker protein (BLNK), which in turn phosphorylates and activates Bruton’s tyrosine kinase (BTK) and phospholipase C  2 (PLC  2). PLC  2 then catalyzes the hydrolysis of phosphatidylinositol-4,5- bisphosphate into diacylglycerol (DAG) and inositol triphosphate (IP 3 ), resulting in increased intracellular calcium levels. The combination of DAG and increased intracellular calcium activates protein kinase C  (PKC  ), which in turn phosphorylates many substrates. These include caspase recruitment domain-containing protein 11 (CARD11), a key signaling adaptor that coordinates a signaling pathway that activates the nuclear factor kappa–light-chain enhancer of activated B cells (NF  B) pathway. CD19 is phosphorylated by the Src-family kinase Lyn during BCR signaling, recruiting phosphatidylinositol 3-kinase (PI3K) to the BCR, which recruits BTK and Akt  The net result of BCR signaling is the activation of the NF  B, PI3K, mitogen-activated protein kinase (MAPK), nuclear factor of activated T cells (NFAT), and Ras pathways, which promote proliferation and survival of normal and malignant B cells  Importantly, malignant B cells can co-opt this pathway to promote their own growth and survival in a variety of ways. Indeed, recent advances have revealed qualitatively different modes of BCR signaling that engage different downstream pathways in B- cell lymphoma  Chronic active BCR signaling engages multiple downstream pathways (MAPK, PI3K, NFAT, and NF  B) and is the type of pathological BCR signaling that drives ABC-DLBCL. In normal B cells, antigen triggers this form of signaling. In ABC-DLBCL, it is thought that BCR signaling is sustained by the constant engagement of the BCR by self-antigens in the tumor microenvironment  Tonic BCR signaling observed in Burkitt’s lymphoma engages the PI3K pathway only and probably is independent of antigen  Qualitative differences in oncogenic BCR signaling in various lymphomas will need to be considered when targeting BCR signaling pathways Reference: Young RM, Staudt LM. Targeting pathological B cell receptor signalling in lymphoid malignancies. Nat Rev Drug Discov. 2013;12: Notes

4  2014 Genentech USA, Inc. All rights reserved. Ras Raf MEK Emerging therapeutic targets in CLL exploit newly recognized features of CLL biology SFK CLL=chronic lymphocytic leukemia; CD 19=B-lymphocyte antigen CD19; SFK=Src-family kinase; Syk=spleen tyrosine kinase; BTK=Bruton’s tyrosine kinase; PI3K=phosphatidylinositol 3-kinase; PDK1=phosphoinositide-dependent kinase-1; Akt=protein kinase B; mTOR=mammalian target of rapamycin; BLNK=B-cell linker; PLCγ2=phospholipase Cγ2; IP3=inositol triphosphate; DAG=diacylglycerol; Ras=rat sarcoma; Raf=rapidly accelerating fibrosarcoma; MEK=mitogen-activated protein kinase kinase; MAPK=mitogen-activated protein kinase; CARD11=caspase recruitment domain-containing protein 11; MALT1=mucosa-associated lymphoid tissue lymphoma translocation protein 1; Bcl10=B-cell lymphoma 10; PKC=protein kinase C; CBM=CARMA1-Bcl10-MALT1 complex; NFκB=nuclear factor kappa–light-chain enhancer of activated B cells.  2014 Genentech USA, Inc. All rights reserved. CD 19 PDK1 Akt PI3K mTOR  Proliferation DAGIP 3 MALT1Bcl-10 CARD11 NFκB PKC   Ca 2+ PLC  2 CBM complex BTK  Cell survival MAPK Syk  Proliferation BLNK 4  Despite many improvements in chemotherapies over the last decade, CLL remains incurable. In contrast to other malignancies, treatment is not always initiated at diagnosis, as chemotherapy is not routinely indicated in patients with early or stable disease 1  Current therapeutic strategies demonstrating efficacy in CLL include several combinations of cytostatic agents and monoclonal antibodies. Much work has been done to optimize these regimens in both first-line and relapsed/refractory settings; however, toxicity and transient responses can narrow treatment options to only stem-cell transplantation for specific groups of patients 1,2  Advances in the understanding of the molecular basis of CLL has presented new targets, thus enabling the development of several promising agents that target a specific signaling lesion or activate host immunity. 3 This novel array of therapeutics is now in clinical trials and has shown preliminary success in treating lymphoma 4  Novel agents target multiple aspects of CLL biology. For example, multiple cell-surface epitopes are available for targeting with monoclonal antibodies. This type of approach has been well established, but steady improvements have been made through innovative antibody design that lead to better response rates 1  In CLL, the PI3K pathway is constitutively activated and dependent on PI3K . Inhibitors of PI3K  in clinical trials have been shown to induce apoptosis of CLL cells. Along those lines, another critical mediator of BCR signaling is BTK. Due to encouraging results in clinical trials, this may soon become an option for CLL patients, especially those with high-risk genetic lesions 5  Another promising strategy involves the intrinsic apoptosis pathway, which becomes unbalanced in CLL, thus favoring survival. Early studies have shown that inhibition of BCL2 prosurvival signals results in tumor lysis within 24 hours. BCL2 inhibitors require further study, but early data suggest a potentially promising role in the treatment of CLL 5  Finally, immunomodulatory drugs appear to target the microenvironment by potentially repairing defective T-cell-to-B-cell synapses 6 References: 1.Smolewski P, Witkowska M, Korycka-Wolowiec A. New insights into biology, prognostic factors, and current therapeutic strategies in chronic lymphocytic leukemia. ISRN Oncol. 2013;2013: Accessed November 8, Cramer P, Hallek M. Prognostic factors in chronic lymphocytic leukemia—what do we need to know? Nat Rev Clin Oncol. 2011;8: Zenz T, Mertens D, Küppers R, Döhner H, Stilgenbauer S. From pathogenesis to treatment of chronic lymphocytic leukaemia. Nat Rev Cancer. 2010;10: Young RM, Staudt LM. Targeting pathological B cell receptor signaling in lymphoid malignancies. Nat Rev Drug Discov. 2013;12: Hallek M. Signaling the end of chronic lymphocytic leukemia: new frontline treatment. Blood. 122: Ramsay AG, Clear AJ, Kelly G, et al. Follicular lymphoma cells induce T-cell immunologic synapse dysfunction that can be repaired with lenalidomide: implications for the tumor microenvironment and immunotherapy. Blood. 2009;114: Notes