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Danielle Hammond, MD

BCL-2 family proteins and conceptualization of BH3 mimetics.

Keying “venetoclax” into this year’s program yields almost 300 hits . Venetoclax has become the cheddar cheese of malignant hematology therapeutics, being “sprinkled” onto nearly every investigational drug regimen. The paradoxical beauty of its action lies in precisely targeting a ubiquitous adjudicator of apoptosis, B-cell leukemia/lymphoma-2 (BCL-2). It wasn’t until Dr. David Vaux and colleagues’ 1988 Nature article about the unexpected role of BCL-2,1 the first inhibitor of apoptosis to be discovered in any species, that it was appreciated that oncogenesis could be driven by resistance to apoptosis rather than excessive proliferation. It only seems fitting that “Die Hard,” a fictional American action film in which the protagonist thwarts a terrorist hijacking of a skyscraper, also debuted in 1988. The Ham-Wasserman lecture delivered this year by Dr. Andrew Roberts of the Walter and Eliza Institute of Australia, captured how the discovery of the antiapoptotic role of BCL-2 has translated over the past 30 years to the development of venetoclax, now one of the most effective therapies available for both chronic lymphocytic leukemia/small cell lymphoma (CLL/SLL) and acute myeloid leukemia (AML). This review article aims to provide a foundation for this remarkable story.

(BCL-2) Family Matters

Apoptosis is a form of controlled cellular demolition that can be triggered by diverse intracellular stresses (e.g., DNA damage, or cytokine or nutrient deprivation). This is governed by a delicate tango of pro- versus antiapoptotic proteins that regulate caspase activation on the mitochondrial outer membrane (MOM). These proteins, collectively termed “BCL-2 family proteins,” share homology in up to four characteristic regions called BCL-2 homology (BH) domains 1 to 4. These proteins belong to one of three functional factions. The antiapoptotic players include the prototype BCL-2, as well as MCL-1, BCL-xL, BCL-w, BCL-B, and BCL2A1/BFL-1. They serve to restrain the faction of proapoptotic effectors whose names sound like a version of the Three Stooges: BAK, BAX, and BOK. These effectors oligomerize to create pores in the MOM that release proapoptotic molecules such as cytochrome c and mitochondria-derived activator caspase, which in turn activate a cascade of proteolytic caspases that results in cellular apoptosis. Ah, but “who will guard the guards?” Enter the third functional faction of proapoptotic intermediaries called “BH3-only” proteins (as they have retained only the shared BH3 domain in their structure). They bind to hydrophobic grooves found on the antiapoptotic proteins, repressing their restraining activity on the Three Stooges. In response to cellular stresses, the BH3-only proteins are mobilized to either a) bind and neutralize their respective antiapoptotic partner or b) directly activate BAK, BAX, or BOK, both mechanisms tipping the balance in favor of apoptosis. Importantly, the various BH3-only proteins (e.g., BAD, BID, BIM, HRK, NOXA, and PUMA) differ in both what type of cellular stress triggers their upregulation and binding affinities for particular antiapoptotic members.

Engineering the Death Star

Many cancers, hematologic and nonhematologic, employ diverse mechanisms to upregulate antiapoptotic, and/or cripple proapoptotic, mediators. One mechanism consists of inactivating mutations in the p53 protein, which under conditions of DNA damage normally induces transcription of two BH3-only proteins, NOXA and PUMA. Deregulation of BCL-2 is a hallmark of CLL and several non-Hodgkin B-cell lymphomas (NHLs). BCL-2 was discovered by virtue of the translocation (14;18) that characterizes follicular lymphoma, which results in the constitutive expression of BCL-2, now transposed downstream of the immunoglobulin heavy-chain promoter region. Deletion of the 13q14.3 locus is common in CLL. This leads to loss of two genes in that region that encode microRNAs (miR-15a and miR-16-1), both of which negatively regulate BCL-2 at the post-transcriptional level, resulting in increased BCL-2 expression. Mechanisms that upregulate antiapoptotic members other than BCL-2 are less well characterized.

Initial attempts to pharmacologically inhibit BCL-2 and other antiapoptotic proteins were unsuccessful. Oblimersen sodium, designed to bind to and target BCL-2 messenger RNA for degradation, was the first BCL-2 inhibitor tested. However, a randomized trial combining the drug with chemoimmunotherapy in relapsed or refractory (R/R) CLL did not show added benefit.2 Obatoclax, the first “BH3 mimetic” to be clinically tested, worked by inhabiting the BH3-binding groove of several of the antiapoptotic proteins, freeing bound proapoptotic effectors and displacing bound BH3-only proteins, shifting the balance toward induction of apoptosis. However, both modest clinical activity and neurologic toxicity observed in early-phase trials in CLL and NHLs ended further development of Obatoclax.3-6 Navitoclax (ABT-263), the subsequent early-generation BH3 mimetic, displayed higher binding affinity for target antiapoptotic proteins and improved clinical activity. However, its dose-limiting toxicity was thrombocytopenia due to on-target BCL-xL inhibition, leading to the apoptotic death of platelets.7 Venetoclax (ABT-199) was the re-engineered product of navitoclax created to be a selective BCL-2 inhibitor, thus sparing platelet survival.8

Venetoclax in CLL

Preclinical work identified CLL/SLL to be highly sensitive to BCL-2 inhibition. Dr. Roberts and colleagues led the first in-human, early-phase studies9,10 of oral venetoclax in R/R CLL/SLL beginning in 2011. Once again demonstrating that there can be too much of a good thing, the first few patients experienced astonishingly rapid tumor kill, resulting in laboratory tumor lysis syndrome within hours of their first dose. This led to lowering the starting dose to 50 mg and an extended weekly dose ramp-up schedule. Despite this, two patients suddenly died (one who received venetoclax in combination with rituximab) and a third experienced acute renal failure necessitating hemodialysis, all secondary to severe clinical tumor lysis syndrome. These heart-wrenching tragedies led to the stringent administration guidelines now followed in clinical practice, which include dosing beginning at 20 mg daily with a weekly ramp-up over five weeks, to a final daily target dose of 400 mg. Two phase II studies of venetoclax monotherapy in patients with R/R CLL (limited to the cases with deletion [17p]11 or previously R/R to ibrutinib or idelalisib,12 respectively) demonstrated impressive overall response rates of 70 to 80 percent and an identical one-year progression-free survival (PFS) of 72 percent. This was the basis of the initial U.S. Food and Drug Administration (FDA) approval of venetoclax in April 2016, as monotherapy for the treatment of R/R disease with deletion (17p). In the MURANO phase III study13 in which patients with R/R CLL received either fixed-duration venetoclax-rituximab versus six cycles of bendamustine-rituximab, the venetoclax-rituximab arm demonstrated a significantly higher two-year PFS (85% vs. 36%) and undetectable measurable residual disease rate (MRD-U, <1 CLL cell in 10,000 leukocytes). However, the prognostic significance of MRD-U using regimens other than fludarabine-cyclophosphamide-rituximab chemoimmunotherapy has not yet been validated. This regimen of venetoclax-rituximab was FDA approved in June 2018 for previously treated CLL irrespective of deletion (17p) status. The phase III German CLL14 study,14 which compared frontline fixed-duration venetoclax-obinutuzumab to clorambucil-obinutuzumab in patients with coexisting comorbidities (presumably making traditional chemoimmunotherapy less desirable), demonstrated superior two-year PFS (88% vs. 64%) and MRD-U rates, with improved PFS being directionally maintained across traditional prognostic subgroups per TP53 and immunoglobulin heavy-chain rearrangement status. This was the basis of the most recent May 2019 FDA approval for venetoclax in treatment-naïve cases of CLL/SLL. Given how effective ibrutinib and other Bruton’s tyrosine kinase inhibitors have also proven in CLL, the field is suffering a poverty of wealth that King Midas knew well. There are several ongoing studies evaluating various permutations of combination and sequential therapy with venetoclax and Bruton’s tyrosine kinase inhibitors.

It is worth discussing why in the treatment of follicular lymphoma, which has similarly high BCL-2 levels, venetoclax is relatively impotent. Constitutive BCL-2 protein expression levels are not the best predictor of sensitivity to venetoclax. There are two additional predictive factors to consider. One is the critical concept of “apoptotic priming” in which certain tumors undergo cellular stresses inherent to their process of malignant transformation, which upregulate their BH3-only faction, driving concomitant upregulation of antiapoptotic proteins to buffer this. Such tumors are thus more dependent on the high-wire balancing act between the various BCL-2 family proteins, leaving them vulnerable to BH3-mimetics and other therapies disrupting the antiapoptotic machinery. The occupational status of BCL-2 and other antiapoptotic members can now be tested for research purposes using a method called BH3 profiling.15 “Induced priming” can alternatively occur following exposure to cytotoxic chemotherapy, providing the rationale for venetoclax combinations. The second factor predictive of sensitivity to venetoclax is that different tumor types are preferentially addicted to particular antiapoptotic proteins. Follicular lymphoma is thought to be less BCL-2 dependent, and more MCL-1 and BCL-xL dependent, for example.16

Venetoclax in AML

Venetoclax for AML had an inauspicious start. An early-phase study of venetoclax monotherapy in patients with R/R AML demonstrated an overall response rates of only 19 percent.17 This threatened to halt further study in AML. However, physician-scientists like Drs. Marina Konopleva and Tony Letai appealed to the concept of induced apoptotic priming to advocate for proceeding with the study of venetoclax combinations that showed synergy in the preclinical setting. Two phase Ib/II studies launched in parallel, combining venetoclax with either a hypomethylating agent or low-dose cytarabine in older patients unsuitable to receive intensive induction chemotherapy. Encouragingly, combined complete response (CR) or complete response with incomplete hematology recovery (CRi) rates were 67 percent and 54 percent, respectively, well exceeding historical response rates with hypomethylating agent or low-dose cytarabine monotherapy. This translated to a median overall survival (OS) of 17.5 months and 10.5 months, representing a significant improvement compared with historical cohorts. Furthermore, NPM1– or IDH1/2-mutated subgroups displayed notably high response rates of approximately 90 percent and 70 percent, respectively. This work has since cumulated in the VIALE-A18 and VIALE-C19 phase III trials. In the VIALE-A study, venetoclax-azacitidine significantly reduced the risk of death by 34 percent compared to azacitidine alone (median OS, 14.7 vs. 9.6 months). The VIALE-C study did not demonstrate an OS benefit, but it did show improved CR rates (27% vs. 7.4%) and durability (11 vs. 8 months). These data were the basis of the October 2020 FDA approval of these venetoclax combinations for the frontline treatment of patients 75 years years or older or who have comorbidities that preclude use of intensive induction chemotherapy. When asked about these recently approved venetoclax combinations in AML, Dr. Konopleva reflected, “It is hard for me to express the excitement, satisfaction, and hope for cure in at least a subset of our patients … I am extremely humbled that in close collaboration with my friend and colleague, Dr. Tony Letai, we stood at the very beginning of this journey, demonstrating in the lab that [venetoclax] is a highly potent therapeutic tool in AML therapy… The overall safety of the compound and its ability to potentiate almost any type of chemotherapy or targeted therapy will eventually make it to be part of multiple therapeutic regimens not only in AML and CLL, but in other hematologic malignancies such as ALL, blastic plasmacytoid dendritic cell neoplasm, and others.”

Moving Forward

In CLL and NHL models, induced upregulation of BCL-xL can drive acquired resistance.20 Acquired G101V mutations interfering with the binding of venetoclax to BCL-2 have also been demonstrated in patients with CLL on continuous venetoclax therapy,21 akin to the ABL1 kinase domain resistance mutations which can emerge in chronic myeloid leukemia treated with tyrosine kinase inhibitors. BCL-2 mutations have yet to be similarly documented in cases of AML treated with venetoclax regimens. However, BH3 profiling has demonstrated gradual switching from dependence on BCL-2 to alternative antiapoptotic proteins, especially MCL-1, under the selective pressure of venetoclax therapy in AML.22 Concurrent BCL-2 and MCL-1 inhibition is therefore an aspirational therapeutic strategy, with a handful of candidate MCL-1 inhibitors already undergoing clinical testing, including in combination with venetoclax.

Dr. Konopleva summarizes the story best, “Engaging apoptosis pathways with BH3 mimetics is a tremendous success of translational and clinical medicine that rapidly became one of the key therapeutic tools in our battle against cancer and leukemias.” Yippee Ki Yay!

 

What’s in a Name?

The Ham-Wasserman Lecture is named in honor of two past ASH presidents, the late Drs. Thomas Hale Ham (1905-1987) and Louis R. Wasserman (1911-1999), both transformative figures in American hematology.

Indeed, Dr. Ham is the eponym of the Ham test ¾ historically used in the diagnosis of paroxysmal nocturnal hemoglobinuria. While his research interests included hemolytic anemias and the applications of erythroid sedimentation rate, his arguably more important contribution was spearheading a more compassionate approach to medical education. He was an educator at heart, having graduated from teachers college prior to his BS and MD degrees. As faculty at the Harvard Medical School and later at Case Western Reserve, he advocated for treating medical learners as valued colleagues and persons, implementing curricula that promoted these values. He also co-authored a narrative history of ASH in 1977 that can be found on the ASH website.

Dr. Wasserman, who was associated with Mount Sinai Hospital in New York City for more than 60 years, led the Polycythemia Vera Study Group (PVSG), an international effort to establish diagnostic criteria, the natural history, and effective therapies for polycythemia (rubra) vera. He also served as vice president of the International Society of Hematology. In recognition of these international efforts, the Ham-Wasserman lecture is traditionally delivered by an individual from outside the United States who has made a major contribution to their respective area in hematology.

Excerpts From Yesterday’s Ham-Wasserman Lecture
  • “Unlike other oncogenes known to that point, BCL-2 did not regulate proliferation or drive growth of tumors, rather it regulated apoptosis. Overexpression of BCL-2 protected cells from death in the face of insults that would normally cause them to die … That discovery had led to an explosion of investigations all around the world into apoptosis and its relationship to cancer.”
  • “When we took venetoclax into patients with CLL … we actually saw very dramatic changes after a single dose of drug, and changes within six to eight hours.”
  • “With cells somewhat sensitive [to BCL-2 ] … rather than being the bullseye which it is in CLL … by inhibiting it we set up a wave of stress on the cell that ultimately may hit the bullseye and kill the cell.”
  • When asked about sequencing versus combination therapy approaches: “Philosophically, I am interested in curing people with currently incurable diseases, and the history of hematology would say that you are going need to use combinations, and you are going to need to use them mostly the first time… I come from the camp that says ‘let’s combine these drugs, let’s aim for CRs with MRD negativity’ as surrogates for long-term disease-free survival.”

— Dr. Andrew Roberts

  1. Vaux DL, Cory S, Adams JM. Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-myc to immortalize pre-B cells. Nature. 1988;335:440-442.
  2. O’Brien S, Moore JO, Boyd TE, et al. 5-year survival in patients with relapsed or refractory chronic lymphocytic leukemia in a randomized, phase III trial of fludarabine plus cyclophosphamide with or without oblimersen. J Clin Oncol. 2009;27:5208-5212.
  3. Goy A, Hernandez-Ilzaliturri FJ, Kahl B, et al. A phase I/II study of the pan Bcl-2 inhibitor obatoclax mesylate plus bortezomib for relapsed or refractory mantle cell lymphoma. Leuk Lymphoma. 2014;55:2761-2768.
  4. Goy A, Berger M, Ford P, et al. Sequential single-agent obatoclax mesylate (GX15-070MS) followed by combination with rituximab in patients with previously untreated follicular lymphoma. Leuk Lymphoma. 2014;55:2932-2934.
  5. O’Brien SM, Claxton DF, Crump M, et al. Phase I study of obatoclax mesylate (GX15-070), a small molecule pan-Bcl-2 family antagonist, in patients with advanced chronic lymphocytic leukemia. Blood. 2009;113:299-305.
  6. Brown JR, Tesar B, Yu L, et al. Obatoclax in combination with fludarabine and rituximab is well-tolerated and shows promising clinical activity in relapsed chronic lymphocytic leukemia. Leuk Lymphoma. 2015;56:3336-3342.
  7. Vogler M, Hamali HA, Sun XM, et al. BCL2/BCL-X(L) inhibition induces apoptosis, disrupts cellular calcium homeostasis, and prevents platelet activation. Blood. 2011;117:7145-7154.
  8. Souers AJ, Leverson JD, Boghaert ER, et al. ABT-199, a potent and selective BCL-2 inhibitor, achieves antitumor activity while sparing platelets. Nat Med. 2013;19:202-208.
  9. Roberts AW, Davids MS, Pagel JM, et al. Targeting BCL2 with venetoclax in relapsed chronic lymphocytic leukemia. N Engl J Med. 2016;374:311-322.
  10. Seymour JF, Ma S, Brander DM, et al. Venetoclax plus rituximab in relapsed or refractory chronic lymphocytic leukaemia: a phase 1b study. Lancet Oncol. 2017;18:230-240.
  11. Stilgenbauer S, Eichhorst B, Schetelig J, et al. Venetoclax in relapsed or refractory chronic lymphocytic leukaemia with 17p deletion: a multicentre, open-label, phase 2 study. Lancet Oncol. 2016;17:768-778.
  12. Jones J, Choi MY, Mato AR, et al. Venetoclax (VEN) monotherapy for patients with chronic lymphocytic leukemia (CLL) who relapsed after or were refractory to ibrutinib or idelalisib. Blood. 2016;128:637.
  13. Seymour JF, Kipps TJ, Eichhorst B, et al. Venetoclax-rituximab in relapsed or refractory chronic lymphocytic leukemia. N Engl J Med. 2018;378:1107-1120.
  14. Fischer K, Al-Sawaf O, Bahlo J, et al. Venetoclax and obinutuzumab in patients with CLL and coexisting conditions. N Engl J Med. 2019;380:2225-2236.
  15. Certo M, Del Gaizo Moore V, Nishino M, et al. Mitochondria primed by death signals determine cellular addiction to antiapoptotic BCL-2 family members. Cancer Cell. 2006;9:351-365.
  16. Roberts AW, Huang DCS. Targeting BCL2 with BH3 mimetics: Basic science and clinical application of venetoclax in chronic lymphocytic leukemia and related B cell malignancies. Clin Pharmacol Ther. 2017;101:89-98.
  17. Konopleva M, Pollyea DA, Potluri J, et al. Efficacy and biological correlates of response in a phase II study of venetoclax monotherapy in patients with acute myelogenous leukemia. Cancer Discov. 2016;6:1106-1117.
  18. DiNardo CD, Jonas BA, Pullarkat V, et al. Azacitidine and venetoclax in previously untreated acute myeloid leukemia. N Engl J Med. 2020;383:617-629.
  19. Wei AH, Montesinos P, Ivanov V, et al. Venetoclax plus LDAC for newly diagnosed AML ineligible for intensive chemotherapy: a phase 3 randomized placebo-controlled trial. Blood. 2020;135:2137-2145.
  20. Chiron D, Bellanger C, Papin A, et al. Rational targeted therapies to overcome microenvironment-dependent expansion of mantle cell lymphoma. Blood. 2016;128:2808-2818.
  21. Tausch E, Close W, Dolnik A, et al. Venetoclax resistance and acquired BCL2 mutations in chronic lymphocytic leukemia. Haematologica. 2019;104:e434-e437.
  22. Teh TC, Nguyen NY, Moujalled DM, et al. Enhancing venetoclax activity in acute myeloid leukemia by co-targeting MCL1. Leukemia. 2018;32:303-312.

Dr. Hammond indicated no relevant conflicts of interest.

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